Nucleic acids encoding a human glycine transporter

ABSTRACT

The invention provides nucleic acids and proteins derived from the sequence of the human GlyT-2 transporter of the amino acid glycine.

This application is related to the following co-pending applications:"Glycine Transporter-Transfected Cells and Uses Thereof," Ser. No.08/655,836, filed May 31, 1996; "Pharmaceutical For Treatment OfNeurological And Neuropsychiatric Disorders," Ser. No. 08/656,063, filedMay 31, 1996, now a provisional application "Pharmaceutical ForTreatment of Neuropsychiatric Disorders," Ser. No. 08/655,912, filed May31, 1996, now a provisional application and "Pharmaceutical For TreatingOf Neurological and Neuropsychiatric Disorders," Ser. No. 08/655,847,filed May 31, 1996, now a provisional application

The present invention relates to nucleic acid encoding the "GlyT-2"member of the family of human glycine transporters, to the isolatedprotein encoded by the nucleic acid, and to the field of drug discovery.

Synaptic transmission is a complex form of intercellular communicationthat involves a considerable array of specialized structures in both thepre- and post-synaptic neuron. High-affinity neurotransmittertransporters are one such component, located on the pre-synapticterminal and surrounding glial cells (Kanner and Schuldiner, CRCCritical Reviews in Biochemistry 22: 1032, 1987). Transporters sequesterneurotransmitter from the synapse, thereby regulating the concentrationof neurotransmitter in the synapse, as well as its duration in thesynapse, which together influence the magnitude of synaptictransmission. By preventing the spread of transmitter to neighboringsynapses, transporters maintain the fidelity of synaptic transmission.Further, by sequestering released transmitter into the presynapticterminal, transporters allow for transmitter reutilization.

Neurotransmitter transport is dependent on extracellular sodium and thevoltage difference across the membrane; under conditions of intenseneuronal firing, as for example during a seizure, transporters canfunction in reverse, releasing neurotransmitter in a calcium-independentnon-exocytotic manner (Attwell et al., Neuron 11: 401-407, 1993).Pharmacologic modulation of neurotransmitter transporters thus providesa means for modifying synaptic activity, which provides useful therapyfor the treatment of neurological and psychiatric disturbances.

The amino acid glycine is a major neurotransmitter in the mammaliannervous system, functioning at both inhibitory and excitatory synapses.By nervous system, both the central and peripheral portions of thenervous system are intended. The distinct inhibitory and excitatoryfunctions of glycine are mediated by two different types of receptor,each of which is associated with a different class of glycinetransporter. The inhibitory actions of glycine are mediated by glycinereceptors that are sensitive to the convulsant alkaloid, strychnine, andare thus referred to as "strychnine-sensitive". Such receptors containan intrinsic chloride channel that is opened upon binding of glycine tothe receptor; by increasing chloride conductance, the threshold forfiring of an action potential is increased. Strychnine-sensitive glycinereceptors are found predominantly in the spinal cord and brainstem, andpharmacological agents that enhance the activation of such receptorswill thus increase inhibitory neurotransmission in these regions.

Glycine functions in excitatory transmission by modulating the actionsof glutamate, the major excitatory neurotransmitter in the centralnervous system. See Johnson and Ascher, Nature 325: 529-531, 1987;Fletcher et al., Glycine Transmission Otterson and Storm-Mathisen,

eds., 1990, pp. 193-219. Specifically, glycine is an obligatoryco-agonist at the class of glutamate receptor termedN-methyl-D-aspartate (NMDA) receptor. Activation of NMDA receptors on aneuron increases sodium and calcium conductance, which depolarizes theneuron, thereby increasing the likelihood that the neuron will fire anaction potential. NMDA receptors are widely distributed throughout thebrain, with a particularly high density in the cerebral cortex andhippocampal formation.

Molecular cloning has revealed the existence in mammalian brains of twoclasses of glycine transporters, termed GlyT-1 and GlyT-2. GlyT-1 isfound predominantly in the forebrain, and its distribution correspondsto that of glutamatergic pathways and NMDA receptors (Smith, et al.,Neuron 8: 927-935, 1992). The distribution of GlyT-2 differs; thistransporter is found predominantly in the brain stem and spinal cord,and its distribution corresponds closely to that of strychnine-sensitiveglycine receptors. Liu et al., J. Biol. Chem. 268: 22802-22808, 1993;Jursky and Nelson, J. Neurochem. 64: 1026-1033, 1995. These observationsare consistent with the view that, by regulating the synaptic levels ofglycine, GlyT-1 and GlyT-2 preferentially influence the activity of NMDAreceptors and strychnine-sensitive glycine receptors, respectively.

Sequence comparisons of GlyT-1 and GlyT-2 have revealed that theseglycine transporters are members of a broader family of sodium-dependentneurotransmitter transporters, including, for example, transportersspecific for γ-amino-n-butyric acid (GABA) and others. Uhl, Trends inNeuroscience 15: 265-268, 1992; Clark and Amara, BioEssays 15: 323-332,1993. Overall, each of these transporters includes 12 putativetransmembrane domains that predominantly contain hydrophobic aminoacids. Comparing rat GlyT-1 to rat GlyT-2, using the Lipman-PearsonFASTA algorithm, reveals a 51% amino acid sequence identity and a 55%nucleic acid sequence identity. Comparison of the sequence of humanGlyT-1 with rat GlyT-2 reveals a 51% amino acid sequence identity and a53-55% nucleic acid sequence identity, with the range of values fornucleic acid sequence identity resulting from the existence of threevariant forms of GlyT-1.

Compounds that inhibit or activate glycine transporters would beexpected to alter receptor function, and provide therapeutic benefits ina variety of disease states. For example, inhibition of GlyT-2 can beused to diminish the activity of neurons having strychnine-sensitiveglycine receptors via increasing synaptic levels of glycine, thusdiminishing the transmission of pain-related (i.e., nociceptive)information in the spinal cord, which has been shown to be mediated bythese receptors. Yaksh, Pain 111-123, 1989. Additionally, enhancinginhibitory glycinergic transmission through strychnine-sensitive glycinereceptors in the spinal cord can be used to decrease musclehyperactivity, which is useful in treating diseases or conditionsassociated with increased muscle contraction, such as spasticity,myoclonus (which relates to rapid muscle spasms), and epilepsy (Truonget al., Movement Disorders 3: 77-87, 1988; Becker, FASEB J. 4:2767-2774, 1990). Spasticity that can be treated via modulation ofglycine receptors is associated with epilepsy, stroke, head trauma,multiple sclerosis, spinal cord injury, dystonia, and other conditionsof illness and injury of the nervous system.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a nucleic acid encoding aglycine transporter having at least about 96% sequence identity with theprotein sequence of SEQ ID 19 or with a sequence corresponding to theprotein sequence of SEQ ID 19 except that it has one or more of thefollowing substitutions (1) Ser¹⁰² to Gly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹to Asn, (4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn, (7)Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ to Ser or (10) Ile⁷³⁵ toVal. Preferably, the sequence identity is at least about 97%, morepreferably at least about 98%, yet more preferably at least about 99%,yet more preferably at least about 99.5%. In an embodiment of theinvention, the sequence identity is 100%. Preferably, the encodedglycine transporter has no more than four amino acid differences in theregion from amino acid 200 to 797 of the protein sequence of SEQ ID 19or of a sequence corresponding to the protein sequence of SEQ ID 19except that it has one of the substitutions described above, morepreferably no more than two such differences.

The invention also provides a vector comprising the nucleic aciddescribed above. In one embodiment, the vector is effective to express aglycine transporter mRNA in at least one of a bacterial cell or aeukaryotic cell. In another embodiment of the invention, the vector iseffective to express the mRNA in at least one of a yeast cell, amammalian cell or an avian cell.

The invention further provides an isolated glycine transporter derivedfrom transformed cells according to the invention, the transportercomprising the amino acid sequence encoded by the above-describednucleic acid or one to two contiguous portions of amino acid sequenceencoded by such a nucleic acid, wherein the protein has glycinetransporter activity and differs in sequence from the aligned segmentsof the rat transporter sequence. The phrase "contiguous sequence," asused herein, refers to uninterupted portions of the relevant referencenucleic acid or amino acid sequence. Preferably, the glycine transporterprotein of the present invention differs in sequence from the alignedsegments of the rat transporter sequence by at least two amino acids,more preferably, at least four amino acids. Preferably, the contiguoussequences comprise at least about 600 amino acids, more preferably atleast about 700 amino acids, more preferably at least about 750 aminoacids. In one embodiment, the transporter protein comprises all of theprotein sequence encoded by the above-described nucleic acid.Preferably, the transporter protein comprises amino acid sequence setforth in the protein sequence of SEQ ID 19 or a sequence correspondingto the protein sequence of SEQ ID 19 except that it has one or more ofthe following substitutions (1) Ser¹⁰² to Gly, (2) Ser¹²⁴ to Phe, (3)Ile²⁷⁹ to Asn, (4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn,(7) Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ to Ser or (10) Ile⁷³⁵to Val, or an amino acid sequence comprising one to two contiguousportions of these sequences. In a preferred embodiment, the inventionprovides a glycine transporter and associated nucleic acids, vectors andmethods, wherein the protein sequence comprises at least one of (1)Gly¹⁰², (2) Phe¹²⁴, (3) Asn²⁷⁹, (4) Gly³⁹³, (5) Asn⁴⁵⁷, (6) Asn⁴⁶³, (7)to Tyr⁶¹⁰ (8) Val⁶¹¹, (9) Ser⁷³³ and (10) Val⁷³⁵. Preferably, thesequence comprises at least two of these amino acid residues, morepreferably at least four, yet more preferably all of these amino acidresidues.

In a second embodiment, the invention also provides a nucleic acidencoding a transporter protein having at least about 99.5% sequenceidentity with all or one to two contiguous portions of the amino acidsequence of SEQ ID 19 or with one to two continous portions of an aminoacid sequence corresponding to the protein sequence of SEQ ID 19 exceptthat it has one or more of the following substitutions (1) Ser¹⁰² toGly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹ to Asn, (4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷to Asn, (6) Asp⁴⁶³ to Asn, (7) Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val, (9)Phe⁷³³ to Ser or (10) Ile⁷³⁵ to Val, wherein the encoded protein hasglycine transporter activity. Preferably, the contiguous sequencescomprise at least about 600 amino acids, more preferably at least about700 amino acids, more preferably at least about 750 amino acids. Theinvention also provides a vector comprising this nucleic acid. In oneembodiment, the vector is effective to express a glycine transportermRNA in at least one of a prokaryotic cell such as a bacterial cell or aeukaryotic cell. In another embodiment of the invention, the vector iseffective to express the mRNA in at least one of a yeast cell, amammalian cell or an avian cell.

The invention additionally provides a cell comprising a firstextrinsically-derived nucleic acid according to the first embodiment ora second extrinsically-derived nucleic acid encoding a transporterprotein having at least about 99.5% sequence identity with one to twocontiguous portions of the protein sequence of SEQ ID 19 or of asequence corresponding to the protein sequence of SEQ ID 19 except thatit has one or more of the following substitutions (1) Ser¹⁰² to Gly, (2)Ser¹²⁴ to Phe, (3) Ile²⁷⁹ to Asn, (4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn,(6) Asp⁴⁶³ to Asn, (7) Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ toSer or (10) Ile⁷³⁵ to Val, wherein the encoded protein has glycinetransporter activity. In one embodiment, the cell expresses a glycinetransporter from the nucleic acid. Preferably, the nucleic acid isfunctionally associated with a promoter that is operative in the cell.In an embodiment of the invention, the promoter is an induciblepromoter.

The invention also provides a method of producing a glycine transportercomprising growing the cells described in the previous paragraph. Thismethod can further comprise at least one of (a) isolating membranes fromsaid cells, which membranes comprise the glycine transporter or (b)extracting a protein fraction from the cells, which fraction comprisesthe glycine transporter.

An embodiment of the invention provides a method for characterizing abioactive agent for treatment of a nervous system disorder or conditionor for identifying bioactive agents for treatment of a nervous systemdisorder or condition, the method comprising (a) providing a first assaycomposition comprising (i) a cell as described above or (ii) an isolatedglycine transporter protein comprising the amino acid sequence encodedby the first or second extrinsically-derived nucleic acids describedabove, (b) contacting the first assay composition with the bioactiveagent or a prospective bioactive agent, and measuring the amount ofglycine transport exhibited by the assay composition. Preferably, themethod further comprises comparing the amount of glycine transportexhibited by the first assay composition with the amount of glycinetransport exhibited by a second such assay composition that is treatedthe same as the first assay composition except that it is not contactedwith the bioactive agent or prospective bioactive agent. The method canbe used for characterizing bioactive agents where the nervous systemdisorder or condition is one of the group consisting of (a) pain,(b)spasticity, (c) myoclonus, (d) muscle spasm, (e) muscle hyperactivityor (f) epilepsy. In a preferred embodiment, the spasticity for which thebioactive agent is characterized is associated with stroke, head trauma,neuronal cell death, multiple sclerosis, spinal cord injury, dystonia,Huntington's disease or amyotrophic lateral sclerosis.

The invention further provides a nucleic acid that hybridizes with thenucleic acid sequence of SEQ ID 18 or with a sequence that varies fromthe nucleic acid sequence of SEQ ID 18 by having one or more of thefollowing substitutions (a) T⁶ to C, (b) A³⁰⁴ to G, (c) C³⁷¹ to T, (d)C⁵⁷¹ to T, (e) T⁸³⁶ to A, (f) A¹¹¹⁶ to G, (g) A¹¹⁷⁷ to G, (h) G¹³⁷¹ toC, (i) G¹³⁸⁷ to A, (j) G¹⁸²⁹ to A, (k) A¹⁸³¹ to G, (I) G²¹⁰³ to A, (m)T²¹⁹⁸ to C, or (n) A²²⁰³ to G, under conditions of sufficient stringencyto exclude hybridizations with (a) the sequence for a rat or mouseGlyT-2 transporter or (b) the sequence for a mammalian GlyT-1transporter. Preferably, the nucleic acid sequence is at least about 18nucleotides in length and has at least about 95% sequence identity witha sequence embedded in the nucleic acid sequence of SEQ ID 18 or asequence that varies from the nucleic acid sequence of SEQ ID 18 byhaving one or more of the following substitutions (a) T⁶ to C, (b) A³⁰⁴to G, (c) C³⁷¹ to T, (d) C⁵⁷¹ to T, (e) T⁸³⁶ to A, (f) A¹¹¹⁶ to G, (g)A¹¹⁷⁷ to G, (h) G¹³⁷¹ to C, (i) G¹³⁸⁷ to A, (j) G¹⁸²⁹ to A, (k) A¹⁸³¹ toG, (I) G²¹⁰³ to A, (m) T²¹⁹⁸ to C, or (n) A²²⁰³ to G. Preferably thenucleic acid sequence is at least about 40 nucleotides in length, morepreferably at least about 100 nucleotides in length. Preferably thenucleic acid sequence has at least about 97% sequence identity with theabove-recited reference sequence, more preferably 99% sequence identity.Preferably, the nucleic acid is a PCR primer and the stringentconditions are PCR conditions effective to amplify a human GlyT-2sequence but not to amplify (a) the sequence for a rat or mouse GlyT-2transporter or (b) the sequence for a mammalian GlyT-1 transporter.

Further, the invention provides a nucleic acid of at least about 18nucleotides in length comprising a contiguous sequence from the codingor noncoding strand of a human GlyT-2 gene or cDNA, wherein thecontiguous sequence has at least 1 sequence difference when comparedwith the rat GlyT-2 gene sequence that aligns with the contiguoussequence. Preferably the nucleic acid sequence is at least about 40nucleotides in length, more preferably at least about 100 nucleotides inlength. Preferably, the contiguous sequence has at least twodifferences, more preferably 3 differences when compared with the ratGlyT-2 gene sequence that aligns with the contiguous sequence.

Still further, the invention provides an antisense molecule comprising acontiguous sequence from a coding or non-coding strand of a human geneor cDNA for GlyT-2 which is effective when administered to a cell,tissue, organ or animal to reduce the expression of GlyT-2 in the cellor in a cell of the tissue, organ or animal, wherein the contiguoussequence has at least 1 sequence difference when compared with the ratGlyT-2 gene sequence that aligns with said contiguous sequence.Preferably, the contiguous sequence has at least two differences, morepreferably 3 differences when compared with the rat GlyT-2 gene sequencethat aligns with the contiguous sequence. The phrase "antisensemolecule," is used herein to refer to a molecule designed to bindgenomic DNA or mRNA to interfere in transcription or translation,including interfering with mRNA stability. Preferably, the contiguoussequence is at least about 15 nucleotides in length. Preferably, thecontiguous stretch is included in the coding or non-coding strand of thenucleic acid sequence of SEQ ID 18 or of a sequence that varies from thenucleic acid sequence of SEQ ID 18 by having one or more of thefollowing substitutions (a) T⁶ to C, (b) A³⁰⁴ to G, (c) C³⁷¹ to T, (d)C⁵⁷¹ to T, (e) T⁸³⁶ to A, (f) A¹¹¹⁶ to G, (g) A¹¹⁷⁷ to G, (h) G¹³⁷¹ toC, (i) G¹³⁸⁷ to A, (j) G¹⁸²⁹ to A, (k) A¹⁸³¹ to G, (I) G²¹⁰³ to A, (m)T²¹⁹⁸ to C, or (n) A²²⁰³ to G. Preferably, the contiguous stretch is inthe coding or non-coding strand of the nucleic acid sequence of SEQ ID18. The invention further provides an expression vector comprising suchan antisense molecule.

The invention also provides a method of reducing GlyT-2 expression in atissue or cell comprising applying to the tissue or cell a GlyT-2expression reducing effective amount of such an antisense molecule or aGlyT-2 expression reducing effective amount of an expression vector forexpressing such an antisense molecule in a tissue or cell.Alternatively, the invention provides a method of treating a nervoussystem disorder or condition comprising applying to a tissue or cell ofa human patient a nervous system disorder or condition treatingeffective amount of such an antisense molecule or a nervous systemdisorder or condition treating effective amount of an expression vectorfor expressing such an antisense molecule in a tissue or cell.

Further, the invention provides a method for detecting whether an animalhas autoimmune antibodies against a glycine transporter, the methodcomprising contacting an antibody preparation from the animal or a bodyfluid from the animal with a polypeptide antigen comprising a glycinetransporter or derived from the glycine transporter. Preferably, thepolypeptide antigen comprises a contiguous sequence encoded by theprotein sequence of SEQ ID 19 or with a sequence corresponding to theprotein sequence of SEQ ID 19 except that it has one or more of thefollowing substitutions (1) Ser¹⁰² to Gly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹to Asn, (4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn, (7)Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ to Ser or (10) Ile⁷³⁵ toVal. Preferably, the contiguous sequence is at least about six aminoacids in length, more preferably at least about ten amino acids inlength, still more preferably at least about fifteen amino acids inlength. In one embodiment of the invention, the peptide antigen isselective for antibodies against either a GlyT-1 transporter or a aGlyT-2 transporter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of several gene fragments of the human GlyT-2gene.

FIG. 2 illustrates which fragment clones were used to construct theclone incorporating the nucleic acid sequence of SEQ ID 20, afull-length clone of the human GlyT-2 gene.

FIGS. 3A-E show a comparison between the nucleic acid sequence of SEQ ID18 and the rat GlyT-2 of SEQ ID NO:26 sequence.

FIGS. 4A-B show a comparison between the amino acid sequence of SEQ ID19 and the rat GlyT-2 of SEQ ID NO:27 sequence.

FIG. 5 shows the measurement of glycine transport in QT-6cells eithertransfected with a human GlyT-2 expression vector or mock transfected.

FIG. 6 shows the concentration dependence of glycine transport in QT-6cells transfected with human GlyT-2.

DEFINITIONS

For the purposes of this application, the following terms shall have themeaning set forth below.

Bioactive agent

A bioactive agent is a substance such as a chemical that can act on acell, virus, tissue, organ or organism, including but not limited todrugs (i.e. pharmaceuticals) to create a change in the functioning ofthe cell, virus, organ or organism. Preferably, the organism is amammal, more preferably a human. In a preferred embodiment of theinvention, the method of identifying bioactive agents of the inventionis applied to organic molecules having molecular weight of about 1500 orless.

extrinsically-derived nucleic acid

Extrinsically-derived nucleic acids are nucleic acids found in a cellthat were introduced into the cell, a parent or ancestor of the cell, ora transgenic animal from which the cell is derived through a recombinanttechnology.

extrinsic promoter functionally associated with a nucleic acid

An extrinsic promoter for a protein-encoding nucleic acid is a promoterdistinct from that used in nature to express a nucleic acid for thatprotein. A promoter is functionally associated with the nucleic acid ifin a cell that is compatable with the promoter the promoter can act toallow the transcription of the nucleic acid.

nucleic acid-specific property

Nucleic acid-specific properties are properties that can be used todistinguish differing nucleic acid molecules. Such properties include,without limitation (i) the nucleotide sequence of all or a portion ofthe molecule, (ii) the size of the molecule, for instance determined byelectrophoresis, (iii) the fragmentation pattern generated by treatmentwith chemicals that fragment nucleic acid or generated by nucleases and(iv) the ability of the molecule or fragments thereof to hybridize withdefined nucleic acid probes.

prospective agent

Prospective agents are substances which are being tested by thescreening method of the invention to determine if they affect glycinetransport.

DETAILED DESCRIPTION OF THE INVENTION

The GlyT-2 nucleic acid sequence of SEQ ID 18 or the correspondingencoded protein sequence of SEQ ID 19, are human relatives of the ratGlyT-2 sequence reported in Liu et al., J. Biol. Chem. 268: 22802-22808,1992. SEQ ID 21, the GlyT-2 protein sequence encoded by the nucleic acidsequence of SEQ ID 19, differs from the amino acid sequence of SEQ ID19, most likely reflecting variant forms of human GlyT-2. Additionalsequences set forth in SEQ IDs 1-25 reflect still further variations.These variations primarily arise from the use of cDNA from pooled mRNAfor several donors to generate the clones. In total, the various humanGlyT-2 -derived nucleic acids that have been isolated reveal thefollowing sequence variations:

    ______________________________________                           Corresponding                   Encoded Amino Amino Acid in    Nucleotide variations                   Acid Variations                                 Rat    ______________________________________    AT.sup.6 T (from SEQ ID 18) to                   NONE (Asp.sup.2  to Asp)                                 Asp    ACT (from SEQ ID 3)    A.sup.304 GC (from SEQ ID 18)                   Ser.sup.102  to Gly                                 Ser    to GGC (from SEQ ID 20)    TT.sup.371 T (from SEQ ID 20) to                   Phe.sup.124  to Ser                                 Ala    TCT (from SEQ ID 18)    C.sup.571 GA (from SEQ ID 18)                   Arg.sup.191  to STOP                                 Arg    to TGA (from SEQ ID 7)    AT.sup.836 C (from SEQ ID 18) to                   lle.sup.279  to Asn                                 lle    AAC (from SEQ ID 20)    GAG.sup.1116  (from SEQ ID 20)                   NONE (Glu.sup.372  to Glu)                                 Glu    to GAA (from SEQ ID 18)    G.sup.1177 GG (from SEQ ID 5) to                   Gly.sup.393  to Arg                                 Arg    AGG (from SEQ ID 18)    AAC.sup.1371 (from SEQ ID 10)                   Asn.sup.457  to Lys                                 Lys    to AAG (from SEQ ID 18)    G.sup.1387 AT (from SEQ ID 18)                   Asp.sup.463  to Asn                                 Asp    to AAT (from SEQ ID 12)    TG.sup.1829 C (from SEQ ID 18)                   Cys.sup.610  to Tyr                                 Cys    to TAC (from SEQ ID 22)    A.sup.1831 TT (from SEQ ID 18)                   lle.sup.611  to Val                                 lle    to GTT (from SEQ ID 20)    GAG.sup.2103  (from SEQ ID 18)                   NONE (Glu.sup.701  to Glu)                                 Glu    to GAA (from SEQ ID 24)    TT.sup.2198 T (from SEQ ID 18)                   Phe.sup.733  to Ser                                 Phe    to TCT (from SEQ ID 24)    A.sup.2203 TA (from SEQ ID 18)                   lle.sup.735  to Val                                 lle    to GTA (from SEQ ID 22)    ______________________________________

Irrespective of the source of this variation, the point variations inpeptide sequence, excepting the insertion of the stop codon, arebelieved not to adversely affect the functioning of GlyT-2. The GlyT-2protein sequence of SEQ ID 19 is most preferred.

The above-described variations primarily reflect sequence variationsbetween human individuals. The material used to generate the nucleicacid sequences described above comprised pools from either twenty-six orninety-two individuals, depending on the particular nucleic acidsequence. The use of pooled source material, together with theprevalence of silent or conservative substitutions, support theconclusion that the variations are reflective of human-derivedvariations rather than mutations generated by the amplificationreactions.

The relationship between the human nucleotide sequence of SEQ ID 18 andthe rat nucleotide sequence for GlyT-2, and between the proteinsequences that they encode, is as set forth in the tables below. Therelatedness values set forth in these tables was determined using theFASTA computer program described by Pearson and Lipman, Proc. Natl.Acad. Sci. USA 85: 2444-2448, 1988.

    ______________________________________    Nucleotide Sequence    (numbered as in SEQ ID 18)                       Percent Identity    ______________________________________    nt 1-2397 (whole sequence)                       89     nt 1-600          82.5    nt 60-170          78    nt 600-2397        91.2    ______________________________________

    ______________________________________    Amino Acid Sequence    (numbered as in SEQ ID 19)                       Percent Identity    ______________________________________    aa 1-797           94.4    aa 1-150           77.1    aa 1-200           80.3    aa 150-797         98.5    aa 200-797         99.2    ______________________________________

Nucleic Acid--encoding glycine transporter

To construct non-naturally occurring glycine transporter-encodingnucleic acids, the native sequences can be used as a starting point andmodified to suit particular needs. For instance, the sequences can bemutated to incorporate useful restriction sites. See Maniatis et al.Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989).Such restriction sites can be used to create "cassettes", or regions ofnucleic acid sequence that are facilely substituted using restrictionenzymes and ligation reactions. The cassettes can be used to substitutesynthetic sequences encoding mutated glycine transporter amino acidsequences. Alternatively, the glycine transporter-encoding sequence canbe substantially or fully synthetic. See, for example, Goeddel et al.,Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For recombinantexpression purposes, codon usage preferences for the organism in whichsuch a nucleic acid is to be expressed are advantageously considered indesigning a synthetic glycine transporter-encoding nucleic acid. Forexample, a nucleic acid sequence incorporating prokaryotic codonpreferences can be designed from a mammalian-derived sequence using asoftware program such as Oligo-4, available from National Biosciences,Inc. (Plymouth, Minn).

The nucleic acid sequence embodiments of the invention are preferablydeoxyribonucleic acid sequences, preferably double-strandeddeoxyribonucleic acid sequences. However, they can also be ribonucleicacid sequences.

Numerous methods are known to delete sequence from or mutate nucleicacid sequences that encode a protein and to confirm the function of theproteins encoded by these deleted or mutated sequences. Accordingly, theinvention also relates to a mutated or deleted version of a humannucleic acid sequence that encodes a protein that retains the ability tobind specifically to glycine and to transport glycine across a membrane.These analogs can have N-terminal, C-terminal or internal deletions, solong as GlyT-2 function is retained. The remaining human GlyT-2 proteinsequence will preferably have no more than about 4 amino acidvariations, preferably no more than 2 amino acid variations, morepreferably no more than 1 amino acid variation, relative to the proteinsequence of SEQ ID 19 or with a sequence corresponding to the proteinsequence of SEQ ID 19 except that it has one or more of the followingsubstitutions (1) Ser¹⁰² to Gly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹ to Asn,(4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn, (7) Cys⁶¹⁰ toTyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ to Ser or (10) Ile⁷³⁵ to Val. Morepreferably, the variations are relative to the protein sequence of SEQID 18. The point variations are preferably conservative pointvariations. Preferably, the analogs will have at least about 96%sequence identity, preferably at least about 97%, more preferably atleast about 98%, still more preferably at least about 99%, yet stillmore preferably at least about 99.5%, to the protein sequence of SEQ ID19 or with a sequence corresponding to the protein sequence of SEQ ID 19except that it has one or more of the following substitutions (1) Ser¹⁰²to Gly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹ to Asn, (4) Arg³⁹³ to Gly, (5)Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn, (7) Cys⁶¹⁰ to Tyr, (8) Ile⁶¹¹ to Val,(9) Phe⁷³³ to Ser or (10) Ile⁷³⁵ to Val. More preferably, the variationsare relative to the protein sequence of SEQ ID 18.

Mutational and deletional approaches can be applied to all of thenucleic acid sequences of the invention that express human GlyT-2proteins. As discussed above, conservative mutations are preferred. Suchconservative mutations include mutations that switch one amino acid foranother within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Gluand Gln;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

A preferred listing of conservative variations is the following:

    ______________________________________    Original Residue      Variation    ______________________________________    Ala                   Gly, Ser    Arg                   Lys    Asn                   Gln, His    Asp                   Glu    Cys                   Ser    Gln                   Asn    Glu                   Asp    Gly                   Ala, Pro    His                   Asn, Gln    lle                   Leu, Val    Leu                   lle, Val    Lys                   Arg, Gln, Glu    Met                   Leu, Tyr, lle    Phe                   Met, Leu, Tyr    Ser                   Thr    Thr                   Ser    Trp                   Tyr    Tyr                   Trp, Phe    Val                   lle, Leu    ______________________________________

The types of variations selected may be based on the analysis of thefrequencies of amino acid variations between homologous proteins ofdifferent species developed by Schulz et al., Principles of ProteinStructure, Springer-Verlag, 1978, on the analyses of structure-formingpotentials developed by Chou and Fasman, Biochemistry 13, 211, 1974 andAdv. Enzymol, 47, 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Natl. Acad.Sci. USA 81, 140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157,105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15, 321-353,1986. All of the references of this paragraph are incorporated herein intheir entirety by reference.

Since the ten identified point variations which create amino acidsubstitutions between the various human GlyT-2 mRNAs identified hereinare believed to be useful in creating functional GlyT-2, proteinsincorporating all combinations of these point variations are believed tobe functional. These variations are within the invention.

For the purposes of this application, a nucleic acid of the invention is"isolated" if it has been separated from other macromolecules of thecell or tissue from which it is derived. Preferably, the compositioncontaining the nucleic acid is at least about 10-fold enriched, withrespect to nucleic acid content, over the composition of the sourcecells. Preferably, the nucleic acid is substantially pure, meaningpurity of at least about 60% w/w with respect to other nucleic acids,more preferably about 80%, still more preferably about 90%, yet morepreferably about 95%.

Hybridization Probes

It will be recognized that many deletional or mutational analogs ofnucleic acid sequences for a glycine transporter will be effectivehybridization probes for glycine transporter-encoding nucleic acid.Accordingly, the invention relates to nucleic acid sequences thathybridize with such glycine transporter-encoding nucleic acid sequencesunder stringent conditions. Preferably, the nucleic acid sequencehybridizes with the nucleic acid sequence of of SEQ ID 18 or with anucleic acid sequence that varies therefrom by one or more of thefollowing substitutions (a) T⁶ to C, (b) A³⁰⁴ to G, (c) C³⁷¹ to T, (d)C⁵⁷¹ to T, (e) T⁸³⁶ to A, (f) A¹¹¹⁶ to G, (g) A¹¹⁷⁷ to G, (h) G¹³⁷¹ toC, (i) G¹³⁸⁷ to A, (j) G¹⁸²⁹ to A, (k) A¹⁸³¹ to G, (I) G²¹⁰³ to A, (m)T²¹⁹⁸ to C, or (n) A²²⁰³ to G.

"Stringent conditions" refers to conditions that allow for thehybridization of substantially related nucleic acid sequences. Forinstance, such conditions will generally allow hybridization of sequencewith at least about 85% sequence identity, preferably with at leastabout 90% sequence identity, more preferably with at least about 95%sequence identity. Such hybridization conditions are described bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, 1989. Hybridization conditions and probes can beadjusted in well-characterized ways to achieve selective hybridizationof human-derived probes.

Nucleic acid molecules that will hybridize to a glycinetransporter-encoding nucleic acid under stringent conditions can beidentified functionally, using methods outlined above, or by using forexample the hybridization rules reviewed in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.

Without limitation, examples of the uses for hybridization probesinclude: histochemical uses such as identifying tissues that express thehuman GlyT-2 transporter; measuring mRNA levels, for instance toidentify a sample's tissue type or to identify cells that expressabnormal levels of glycine transporter; and detecting polymorphisms inthe glycine transporter gene. RNA hybridization procedures are describedin Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold SpringHarbor Press, 1989).

PCR Primers

Rules for designing polymerase chain reaction ("PCR") primers are nowestablished, as reviewed by PCR Protocols, Cold Spring Harbor Press,1991. Degenerate primers, i.e., preparations of primers that areheterogeneous at given sequence locations, can be designed to amplifynucleic acid sequences that are highly homologous to, but not identicalto, a human GlyT-2 nucleic acid. Strategies are now available that allowfor only one of the primers to be required to specifically hybridizewith a known sequence. See, Froman et al., Proc. Natl. Acad. Sci. USA85: 8998, 1988 and Loh et al. Science 243: 217, 1989. For example,appropriate nucleic acid primers can be ligated to the nucleic acidsought to be amplified to provide the hybridization partner for one ofthe primers. In this way, only one of the primers need be based on thesequence of the nucleic acid sought to be amplified.

PCR methods of amplifying nucleic acid will utilize at least twoprimers. One of these primers will be capable of hybridizing to a firststrand of the nucleic acid to be amplified and of priming enzyme-drivennucleic acid synthesis in a first direction. The other will be capableof hybridizing the reciprocal sequence of the first strand (if thesequence to be amplified is single stranded, this sequence willinitially be hypothetical, but will be synthesized in the firstamplification cycle) and of priming nucleic acid synthesis from thatstrand in the direction opposite the first direction and towards thesite of hybridization for the first primer. Conditions for conductingsuch amplifications, particularly under preferred stringenthybridization conditions, are well known. See, for example, PCRProtocols, Cold Spring Harbor Press, 1991.

Vectors

A suitable expression vector is capable of fostering expression of theincluded GlyT-2 encoding DNA in a host cell, which can be eukaryotic,fungal, or prokaryotic. Suitable expression vectors include pRc/CMV(Invitrogen, San Diego, Calif.), pRc/RSV (Invitrogen), pcDNA3(Invitrogen), Zap Express Vector (Stratagene Cloning Systems, LaJolla,Calif.); pBk/CMV or pBk-RSV vectors (Stratagene), Bluescript IISK±Phagemid Vectors (Stratagene), LacSwitch (Stratagene), pMAM and pMAMneo (Clontech, Palo Alto, Calif.), pKSV10 (Pharmacia, Piscataway, N.J.),pCRscript (Stratagene) and pCR2.1 (Invitrogen), among others. Usefulyeast expression systems include, for example, pYEUra3 (Clontech).Useful baculovirus vectors include several viral vectors from Invitrogen(San Diego, Calif.) such as pVL1393, pVL1392, pBluBac2, pBluBacHis A, Bor C, and pbacPAC6 (from Clontech).

Cells

In one embodiment of the invention, the transporter is preferablyexpressed in a mammalian cell line, preferably a transformed cell linewith an established cell culture history. In this embodiment,particularly preferred cell lines include COS-1, COS-7, LM(tk⁻), HeLa,HEK293, CHO, Rat-1 and NIH3T3. Other preferred cells include avian cellssuch as QT-6 cells. Other cells that can be used include insect cellssuch as drosophila cells, fish cells, amphibian cells and reptiliancells.

In another embodiment, the transporter is expressed in a cell line thatis more inexpensively maintained and grown than are mammalian celllines, such as a bacterial cell line or a yeast cell line.

Isolated Glycine Transporter

The invention also provides for the human GlyT-2 proteins encoded by anyof the nucleic acids of the invention preferably in a purity of at leastabout 80% with respect to proteins, preferably 90%, more preferably 95%.The purities are achieved, for example, by applying protein purificationmethods, such as those described below, to a lysate of a recombinantcell according to the invention.

The human GlyT-2 variants of the above paragraphs can be used to createorganisms or cells that produce human GlyT-2 activity. Purificationmethods, including associated molecular biology methods, are describedbelow.

Method of Producing Glycine Transporter

One simplified method of isolating polypeptides synthesized by anorganism under the direction of one of the nucleic acids of theinvention is to recombinantly express a fusion protein wherein thefusion partner is facilely affinity purified. For instance, the fusionpartner can be glutathione S-transferase, which is encoded on commercialexpression vectors (e.g., vector pGEX4T3, available from Pharmacia,Piscataway, N.J.). The fusion protein can then be purified on aglutathione affinity column (for instance, that available fromPharmacia, Piscataway, N.J.). Of course, the recombinant polypeptidescan be affinity purified without such a fusion partner using anappropriate antibody that binds to GlyT-2. Methods of producing suchantibodies are available to those of ordinary skill in light of theample description herein of GlyT-2 expression systems and known antibodyproduction methods. See, for example, Ausubel et al., Short Protocols inMolecular Biology, John Wiley & Sons, New York, 1992. If fusion proteinsare used, the fusion partner can be removed by partial proteolyticdigestion approaches that preferentially attack unstructured regionssuch as the linkers between the fusion partner and GlyT-2. The linkerscan be designed to lack structure, for instance using the rules forsecondary structure forming potential developed, for instance, by Chouand Fasman, Biochemistry 13, 211, 1974 and Chou and Fasman, Adv. inEnzymoL. 47, 45-147, 1978. The linker can also be designed toincorporate protease target amino acids, such as, arginine and lysineresidues, the amino acids that define the sites cleaved by trypsin. Tocreate the linkers, standard synthetic approaches for makingoligonucleotides can be employed together with standard subcloningmethodologies. Other fusion partners besides GST can be used. Proceduresthat utilize eukaryotic cells, particularly mammalian cells, arepreferred since these cells will post-translationally modify the proteinto create molecules highly similar to or functionally identical tonative proteins.

Additional purification techniques can be applied, including withoutlimitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala,Sweden), HPLC (e.g., using gel filtration, reverse-phase or mildlyhydrophobic columns), gel filtration, differential precipitation (forinstance, "salting out" precipitations), ion-exchange chromatography andaffinity chromatography.

Because GlyT-2 is a membrane protein, which by analogy to relatedtransporter proteins is believed to have twelve transmembrane sequences,isolation methods will often utilize detergents, generally non-ionicdetergents, to maintain the appropriate secondary and tertiary structureof the protein. See, for example, Lopez-Corcuera et al., J. Biol Chem.266: 24809-24814, 1991. For a description of methods for re-integratinga solubilized transporter into a membrane, see Lopez-Corcuera et al., J.Biol. Chem. 266: 24809-24814, 1991.

The isolation of GlyT-2 can comprise isolating membranes from cells thathave been transformed to express GlyT-2. Preferably, such cells expressGlyT-2 in sufficient copy number such that the amount of GlyT-2 in amembrane fraction is at least about 10-fold higher than that found incomparable membranes from cells that naturally express GlyT-2, morepreferably the amount is at least about 100-fold higher.

Preferably, the protein is substantially pure, meaning a purity of atleast 60% w/w with respect to other proteins. For the purposes of thisapplication, GlyT-2 is "isolated" if it has been separated from otherproteins or other macromolecules of the cell or tissue from which it isderived. Preferably, the composition containing GlyT-2 is at least about10-fold enriched, preferably at least about 100-fold, with respect toprotein content, over the composition of the source cells.

Expression of GlyT-2 by RNA Insertion

It will be recognized that human GlyT-2 can be expressed by the simplemethod of inserting mRNA into a cell. RNA for these uses can be preparedby sub-cloning the nucleic acid encoding a protein with GlyT-2 activityinto a vector containing a promoter for high efficiency in vitrotranscription, such as a SP6 or T7 RNA polymerase promoter. RNAproduction from the vector can be conducted, for instance, with themethod described in Ausubel et al., Short Protocols in MolecularBiology, John Wiley & Sons, New York, 1992, pp. 10-63 to 10-65.Insertion of RNA into Xenopus-derived oocytes is described, forinstance, in Liu et al. FEBS Letters 305: 110-114, 1992 and Bannon etal., J. Neurochem. 54: 706-708, 1990.

Alternatively, it will be recognized that human GlyT-2 can be expressedby the simple method of inserting mRNA into an in vitro translationsystem, which can be a membrane-containing translation system.Expression of proteins in vitro is described, for instance, in Ausubelet al., Short Protocols in Molecular Biology, John Wiley & Sons, NewYork, 1992, pp. 10-63 to 10-65. See, also, Guastella et al., Science249: 1303-1306, 1990 (in vitro expression of a transporter). The use ofsubcellular membranous material to produce membrane proteins in vitro isdescribed in Walter and Blobel, Meth. Enzymol. 96: 84, 1983 (for rabbitreticulocyte translation system) and Spiess and Lodish, Cell 44: 177,1986 (for wheat germ translation system).

Method of Characterizing or Identifying agent

A method for the analysis of or screening for a bioactive agent fortreatment of a disease or condition associated with a nervous systemdisorder or condition comprises culturing separately first and secondcells, wherein the first and second cells are preferably of the samespecies, more preferably of the same strain thereof, and comprise anexogenous nucleic acid encoding a glycine transporter as describedherein. The nervous system disorders or conditions for which the agentcan be used for treatment include, but are not limited to, (a) pain, (b)myoclonus, (c) muscle spasm, (d) muscle hyperactivity, (e) epilepsy or(f) spasticity such as that associated with stroke, head trauma,neuronal cell death, multiple sclerosis, spinal cord injury, dystonia,Huntington's disease or amyotrophic lateral sclerosis. In this method,the first cell is contacted with the bioactive agent or a prospectiveagent, which is preferably a compound, such as a peptide or an organiccompound in the presence of glycine, which preferably incorporates aradioisotope, such as ³ H or ¹⁴ C. The contacted first cell is thentested for enhancement or inhibition of glycine transport into the firstcell as compared to glycine transport into the second cell that was notcontacted with the compound (i.e., the control cell). Such analysis orscreening preferably includes activities of finding, learning,discovering, determining, identifying, or ascertaining.

Alternatively, the assay can utilize a composition comprising anisolated GlyT-2 transporter in place of cells. Preferably, suchpreparation of isolated transporter will comprise vesicles of membraneor lipid bilayer, which vesicles have an inside and an outside acrosswhich transport can be measured. See, for example, Kanner, Biochemistry17: 1207-1211, 1978.

A bioactive agent is an enhancer of glycine transport uptake if at theend of the test the amount of intracellular or intravesicle glycine isgreater in the agent-contacted composition than in thenon-agent-contacted composition; conversely, a bioactive agent is aninhibitor of glycine transport if the amount of intracellular orintravesicle glycine is greater in the non-agent-contacted compositionas compared to the other. Preferably, the difference in glycine uptakebetween a tested first composition and a control second composition isat least about two-fold; more preferably, the difference is at leastabout five-fold; most preferably, the difference is at least aboutten-fold or greater.

A bioactive agent that is an inhibitor or an enhancer with respect tothe GlyT-2 transporter may have a neutral or opposite effect withanother glycine transporter, such as one of the GlyT-1 transporters.Preferred bioactive agents have specificity to enhance or inhibit theGlyT-2 transporter and have neutral or negligible effect on otherglycine transporters. Preferably, a bioactive agent has at least anorder of magnitude greater potency, reflected in a concentrationdependent parameter such as the IC₅₀ value, in inhibiting or activatingglycine uptake mediated by the GlyT-2 transporter as compared to itseffect on the second glycine transporter. More preferred agents havegreater potencies of at least about 100-fold for one of the glycinetransporters as compared to the other.

The bioactive agent can be any compound, material, composition, mixture,or chemical, that can be presented to a glycine transporter in a formthat allows for the agent to diffuse so as to contact the transporter.Such bioactive agents include but are not limited to polypeptidespreferably of two up to about 25 amino acids in length, more preferablyfrom two to about ten, yet more preferably from two to about five aminoacids in length. Other suitable bioactive agents in the context of thepresent invention include small organic compounds, preferably ofmolecular weight between about 100 daltons and about 5,000 daltons, andare composed of such functionalities as alkyl, aryl, alkene, alkyne,halo, cyano and other groups, including heteroatoms or not. Such organiccompounds can be carbohydrates, including simple sugars, amino or iminoacids, nucleic acids, steroids, and others. The chemicals tested asprospective agents can be prepared using combinatorial chemicalprocesses known in the art or conventional means for chemical synthesis.Preferably, bioactive agents are useful as drugs for treatment ofnervous system disorders or conditions.

Some compounds that inhibit GlyT-1 or GlyT-2 mediated transport alsobind to the glycine binding site on the strychnine-sensitive receptor,or to the glycine binding site on the NMDA receptor. Such binding to thestrychnine-sensitive receptor can be identified by a binding assaywhereby, for example, radiolabeled strychnine is placed in contact witha preparation of strychnine-sensitive receptors, such as can be preparedfrom a membrane fraction from spinal cord or brain stem tissue. Amembrane fraction can be prepared using conventional means, including,for example, methods of homogenization and centrifugation.

Such binding to the NMDA receptor can be identified by a binding assaywhereby, for example, radiolabeled glycine is placed in contact with apreparation of NMDA receptors, such as can be prepared from a membranefraction from neuronal cells or brain tissue. Grimwood et al., Molec.Pharmacol., 41:923-930, 1992. The NMDA receptors located in suchmembranes are treated using mild detergent, such as about 0.1% to about0.5% saponin, to remove any endogenous glycine or glutamate.

The ligand used in such a binding assay is radiolabeled with anydetectable isotope, such as radioactive isotopes of carbon or hydrogen.Specific binding of the radiolabeled ligand is then determined bysubtracting the radioactivity due to non-specific binding from thatwhich is due to total (i.e., specific and non-specific) binding of theradiolabeled ligand. The radioactivity due to non-specific binding isdetermined by measuring the amount of radiolabel associated with astrychnine-sensitive or NMDA receptor-containing membrane fraction thathas been contacted with both radiolabeled ligand and a significantexcess of non-radiolabeled ligand, such as a 100-fold excess. Theradioactivity due to total binding of the radiolabeled ligand isdetermined by measuring the amount of radiolabel bound to the receptorpreparation in the absence of non-radiolabeled ligand. For the NMDAreceptor, one can also measure binding to the glycine site on thereceptor using labeled analogs of amino acids, such as, for example,dichlorokynurenic acid or L-689,560. See, for example, Grimwood et al.,Molecular Pharmacol, 49: 923-930, 1992.

Functional ion-flux assays are used to measure the effect of compoundsidentified by the present invention in enhancing or inhibiting calciumflux (for NMDA receptor preparations) or chloride flux (forstrychnine-sensitive receptor preparations). This test is performed oncell cultures that have membrane-bound NMDA receptors orstrychininesensitive receptors and glycine transporters. Such cellsinclude neuronal cells generally, including those of the brain stem andspinal cord, and cell lines derived therefrom, and any other cell thathas been induced or transfected to express NMDA receptors orstrychnine-sensitive receptors. Calcium used in such a test is commonlythe ⁴⁵ Ca isotope, although other calcium measuring techniques can beused as well, such as calcium-associated fluorescence, which can befluorescence associated with a calcium chelator, and the like. Chlorideused in such a test usually includes the isotope ³⁶ CI. By whatevermethod the calcium or chloride is monitored, ion flux can be enhanced orinhibited as a result of the discrete addition of a bioactive agent ofthe present invention. An advantage of this system is that it allows oneto monitor the net effect on NMDA receptor or strychnine-sensitivereceptor function of a compound that interacts with both the glycinesite on a receptor and on a glycine transporter.

GlyT-2 inhibitors that are also strychnine-sensitive receptor agonistsact in the above-described indications by increasing glycineconcentrations at the strychnine-sensitive receptor-expressing synapsevia inhibition of the glycine transporter, and via directly enhancingstrychnine-sensitive receptor activity. Glycine transporter inhibitorsthat are also strychnine-sensitive receptor antagonists can nonethelessretain activity in treating these indications, if the increase inglycine due to glycine transport inhibition prevails over thestrychnine-sensitive receptor antagonism. Where the strychnine-sensitivereceptor antagonist activity prevails over the effect of increasedextracellular glycine resulting from inhibition of the glycinetransporter, these compounds are useful in treating conditionsassociated with decreased muscle activity such as myasthenia gravis.

As discussed above, the bioactive agents of the invention can have anumber of pharmacological actions. The relative effectiveness of thecompounds can be assessed in a number of ways, including the following:

1. Comparing the activity mediated through GlyT-1 and GlyT-2transporters. This testing identifies bioactive agents (a) that are moreactive against GlyT-1 transporters and thus more useful in treating orpreventing schizophrenia, increasing cognition and enhancing memory or(b) that are more active against GlyT-2 transporters and thus moreuseful in treating or preventing epilepsy, pain or spasticity.

2. Testing for strychnine-sensitive receptor or NMDA receptor binding.This test establishes whether there is sufficient binding at this siteto warrant further examination of the pharmacological effect of suchbinding.

3. Testing the activity of the compounds in enhancing or diminishing ionfluxes in primary tissue culture, for example chloride ion fluxesmediated by strychnine-sensitive receptors or calcium ion fluxesmediated by NMDA receptors. A bioactive agent that increases ion fluxeither (a) has little or no antagonist activity at thestrychnine-sensitive receptor and should not affect the potentiation ofglycine activity through GlyT-2 transporter inhibition or (b), if markedincreases are observed over results with comparative GlyT-2 inhibitorsthat have little direct interaction with strychnine-sensitive receptors,then the agent is a receptor agonist.

In some cases, the agent analysis method of the invention will be usedto characterize whether a bioactive agent is useful in treating anindication in which NMDA receptors and GlyT-1 transporters areimplicated. In this case, generally, a lower measure of activity withrespect to strychnine-sensitive receptors and GlyT-2 transporters ismore desirable.

Antisense Therapies

One aspect of the present invention is directed to the use of"antisense" nucleic acid to treat neurological indications such as thoseidentified above. The approach involves the use of an antisense moleculedesigned to bind mRNA coding for a GlyT-2, thereby stopping orinhibiting the translation of the mRNA, or to bind to the GlyT-2 gene tointerfere with its transcription. For discussion of the design ofnucleotide sequences that bind genomic DNA to interfere withtranscription, see Helene, Anti-Cancer Drug Design 6, 569, 1991. Oncethe sequence of the mRNA sought to be bound is known, an antisensemolecule can be designed that binds the sense strand by the Watson-Crickbase-pairing rules, forming a duplex structure analogous to the DNAdouble helix. Gene Regulation: Biology of Antisense RNA and DNA, Eriksonand lxzant, eds., Raven Press, New York, 1991; Helene, Anti-Cancer DrugDesign, 6:569 (1991); Crooke, Anti-Cancer Drug Design 6, 609, 1991.

A serious barrier to fully exploiting this antisense technology is theproblem of efficiently introducing into cells a sufficient number ofantisense molecules to effectively interfere with the translation of thetargeted mRNA or the function of DNA. One method that has been employedto overcome this problem is to covalently modify the 5' or the 3' end ofthe antisense polynucleic acid molecule with hydrophobic substituents.These modified nucleic acids generally gain access to the cells interiorwith greater efficiency. See, for example, Boutorin et al., FEBS Lett.23,1382-1390, 1989; Shea et al, Nucleic Acids Res. 18, 3777-3783, 1990.Additionally, the phosphate backbone of the antisense molecules has beenmodified to remove the negative charge for example, Agris et al.,Biochemistry 25, 6268, 1986; Cazenave and Helene in Antisense NucleicAcids and Proteins: Fundamentals and Applications, Mol and Van der Krol,eds., p. 47 et seq., Marcel Dekker, New York, 1991) or the purine orpyrimidine bases have been modified (see, for example, Antisense NucleicAcids and Proteins: Fundamentals and Applications, Mol and Van der Krol,eds., p. 47 et seq., Marcel Dekker, New York, 1991; Milligan et al. inGene Therapy For Neoplastic Diseases, Huber and Laso, eds., p. 228 etseq., New York Academy of Sciences, New York, 1994). Other methods toovercome the cell penetration barrier include incorporating theantisense polynucleic acid sequence into an expression vector that canbe inserted into the cell in low copy number, but which, when in thecell, can direct the cellular machinery to synthesize more substantialamounts of antisense polynucleic molecules. See, for example, Farhood etal., Ann. N.Y. Acad. Sci. 716, 23, 1994. This strategy includes the useof recombinant viruses that have an expression site into which theantisense sequence has been incorporated. See, e.g., Boris-Lawrie andTemin, Ann. N.Y. Acad. Sci., 716:59 (1994). Others have tried toincrease membrane permeability by neutralizing the negative charges onantisense molecules or other nucleic acid molecules with polycations.See, e.g. Wu and Wu, Biochemistry, 27:887-892, 1988; Behr et al., Proc.Natl. Acad Sci U.S.A. 86:6982-6986, 1989.

For gene therapy such as antisense therapy, medical workers often try toincorporate, into one or more cell types of an organism, a DNA vectorcapable of directing the synthesis of a protein missing from the cell oruseful to the cell or organism when expressed in greater amounts. Themethods for introducing DNA to cause a cell to produce a new protein ora greater amount of a protein are called "transfection" methods. See,generally, Neoplastic Diseases, Huber and Lazo, eds., New York Academyof Science, New York, 1994; Feigner, Adv. Drug Deli Rev., 5:163 (1990);McLachlin, et al., Progr. Nucl. Acids Res. Mol. Biol., 38:91 (1990);Karlsson, S. Blood, 78:2481 (1991); Einerhand and Valerio, Curr. Top.Microbiol. Immunol, 177:217-235 (1992); Makdisi et al., Prog. LiverDis., 10:1 (1992); Litzinger and Huang, Biochim. Biophys. Acta, 1113:201(1992); Morsy et al., J.A.M.A., 270:2338 (1993); Dorudi et al., BritishJ. Surgery, 80:566 (1993).

Other general methods of incorporating nucleic acids into cells includecalcium phosphate precipitation of nucleic acid and incubation with thetarget cells (Graham and Van der Eb, Virology, 52:456, 1983),co-incubation of nucleic acid, DEAE-dextran and cells (Sompayrac andDanna, Proc. Natl. Acad. Sci., 12:7575, 1981), electroporation of cellsin the presence of nucleic acid (Potter et al., Proc. Natl. Acad. Sci.,81:7161-7165, 1984), incorporating nucleic acid into virus coats tocreate transfection vehicles (Gitman et al., Proc. Natl. Acad. Sci.U.S.A., 82:7309-7313, 1985) and incubating cells with nucleic acidincorporated into liposomes (Wang and Huang, Proc. Natl. Acad. Sci.,84:7851-7855, 1987). One approach to gene therapy is to incorporate thegene sought to be introduced into the cell into a virus, such as aherpes virus, adenovirus, parvovirus or a retrovirus. See, for instance,Akli et al., Nature Genetics 3, 224, 1993.

The nucleic acid compositions of the invention can be, for example,administered orally, topically, rectally, nasally, vaginally, byinhalation, for example by use of an aerosol, or parenterally,e.g.iintramuscularly, subcutaneously, intraperitoneally,intraventricularly, or intravenously. The nucleic acid compositions canbe administered alone, or they can be combined with apharmaceutically-acceptable carrier or excipient according to standardpharmaceutical practice. For the oral mode of administration, thenucleic acid compositions can be used in the form of tablets, capsules,lozenges, troches, powders, syrups, elixirs, aqueous solutions andsuspensions, and the like. In the case of tablets, carriers that can beused include lactose, sodium citrate and salts of phosphoric acid.Various disintegrants such as starch, and lubricating agents such asmagnesium stearate, sodium lauryl sulfate and talc, are commonly used intablets. For oral administration in capsule form, useful diluents arelactose and high molecular weight polyethylene glycols. When aqueoussuspensions are required for oral use, the nucleic acid compositions canbe combined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring agents can be added. For parenteraladministration, sterile solutions of the conjugate are usually prepared,and the pH of the solutions are suitably adjusted and buffered. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic. For ocular administration, ointmentsor droppable liquids may be delivered by ocular delivery systems knownto the art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. For pulmonary administration,diluents and/or carriers will be selected to be appropriate to allow theformation of an aerosol.

Generally, the nucleic acid compositions will be administered in aneffective amount. For pharmaceutical uses, an effective amount is anamount effective to either (1) reduce the symptoms of the indicationsought to be treated or (2) induce a pharmacological change relevant totreating or preventing the indication sought to be treated.

For viral gene therapy vectors, dosages will generally be from about 1μg to about 1 mg of nucleic acid per kg of body mass. For non-infectivegene therapy vectors, dosages will generally be from about 1 μg to about100 mg of nucleic acid per kg of body mass. Antisense oligonucleotidedosages will generally be from about 1 μg to about 100 mg of nucleicacid per kg of body mass.

Autoimmune Disorders

Autoimmune disorders whereby antibodies are produced against glycinetransporters can be expected to be associated with disease states. Forexample, for the GlyT-2 transporters, such disorders can be expected tobe associated with decreased muscle activity, for instance decreasedmuscle activity that presents much like myasthenia gravis, or to beassociated with decreased pain perception. See, for an example of adisease caused by autoantibodies to a molecule involved inneurotransmission (glutamic acid decarboxylase), Nathan et al., J.Neurosci. Res. 40: 134-137, 1995.

The presence of these antibodies can be measured by establishedimmunological methods using protein sequences obtained from the nucleicacids described herein or the related glycine transporters reportedelsewhere. See, for example, Kim et al., Mol. Pharmacol., 45: 608-617,1994 and Liu et al., J. Biol. Chem. 268: 22802-22808, 1992. Suchimmunological methods are described, for example, in Ausubel et al.,Short Protocols in Molecular Biology, John Wiley & Sons, New York, 1992.

The following examples further illustrate the present invention, but ofcourse, should not be construed as in any way limiting its scope.

EXAMPLE 1

GlyT-2 Cloning

The cDNA encoding human GlyT-2 was generated by Reverse-TranscriptionPCR (RT-PCR) in two steps. In the first step, a degenerate primercorresponding to the rat GlyT-2 nucleotide sequence from 2540 to 2521(5'-GGRTCDATCATRTTYTTRTA SEQ ID NO:28) was used to prime cDNA synthesisfrom human spinal cord poly A mRNA (Clontech, Palo Alto, Calif.). Thenumbering recited herein for the rat sequence is according to thenumbering reported in Liu et al., J. Biol. Chem. 268: 22802-22808, 1992.The following primer pairs were then used in PCR reactions:

Primer A1: 5'-CCNAARGARATGAAYAARCCNCC (SEQ ID NO:29, based on NT 223-245of rat sequence)

Primer A2: 5'-GCNGTGAAGTACACCACTTTNCC (SEQ ID NO:30, based on NT1490-1468 of rat sequence)

Primer B1: 5'-CCNAARGARATGAAYAARCCNCC (SEQ ID NO:29, based on NT 223-245of rat sequence; same primer as Primer A1)

Primer B2: 5'-GGCYTCNGGGTAARCCACRAANGC (SEQ ID NO:31, based on NT1872-1849 of rat sequence)

The designation "R" indicates that the oligonucleotide composition has amixture of adenosine and guanosine at the indicated position; "N" is formixed oligonucleotides with all four base combinations at the indicatedposition; "Y" is for mixtures of cytosine and thymidine; "K" is formixtures of guanosine and thymidine; "D" is for mixtures of adenosine,guanosine and thymidine.

The fragments generated by the A1+A2 primers and by the B1+B2 primerswere separately cloned into pCRscript (Stratagene, La Jolla, Calif.) orpCR2.1 (Invitrogen, San Diego, Calif.), and sequenced from the resultingclones using the AutoRead sequencing kit (Pharmacia, Piscataway, N.J.).Comparison of these sequences to rat GlyT-2 using the Lipman-PearsonFASTA algorithm revealed a 89% identity, confirming that these sequencesencoded human GlyT-2. The A1+A2 primer pair produced clone phG2-1, whichhas the nucleic acid sequence of SEQ ID 5 as its insert. The B1+B2primer pair produced clone phG2-2, which has the nucleic acid sequenceof SEQ ID 7 as its insert.

For the second step, cDNA was synthesized from human spinal cord orcerebellum mRNA (Clontech, Palo Alto, Calif.) using random hexamers(Promega, Madison, Wis.), and additional primers were designed basedupon the sequence of clones phG2-1 and phG2-2 for PCR. The followingprimer pairs were used to amplify the 5' and 3' ends of the human GlyT-2cDNA.

Primer C1: 5'-CGGTTCAATCTGTTGTCCGCATCAGACATG (SEQ ID NO:32, based on NT181-210 of rat sequence)

Primer C2: 5'-GCAGGCTCGCGCGTCCGCTG (SEQ ID NO:33, based on NT 210-191 ofhuman sequence)

Primer D1: 5'-CCCGTATGTCGTACTCGTGATCCTCCTCATCCG (SEQ ID NO:34, based onNT 1284-1316 of human sequence)

Primer D2: 5'-CCNCCRTGNGTDATCATNGGRAANCCC (SEQ ID NO:35, based on NT2087-2061 of rat sequence)

Primer E1: 5'-CCCGTATGTCGTACTCGTGATCCTCCTCATCCG (SEQ ID NO:34, based onNT 1284-1316 of human sequence; same primer as Primer D1)

Primer E2: 5'-CCATCCACACTACTGGAYYARCAYTGNGTNCC (SEQ ID NO:36, based onNT 2624-2593 of rat sequence)

Primer F1: 5'-CAGATTTCCTTCTCTTTATCTGCTGCATGG (SEQ ID NO:37, based on NT1417-1446 of human sequence)

Primer F2: 5'-GGRTCDATCATRTTYTTRTANCKYTCNCC (SEQ ID NO:38, based on NT2540-2512 of rat sequence)

Primer G1: 5'-CCTGCACCAACAGTGCCACAAGC (SEQ ID NO:39, based on NT1517-1539 of human sequence)

Primer G2: 5'-CCATCCACACTACTGGAYYARCAYTGNGTNCC (SEQ ID NO:36, based onNT 2624-2593 of rat sequence; same primer as Primer E2)

Primer H1: 5'-CCAAGTACCTACGCACACACAAGCC (SEQ ID NO:40, based on NT1784-1808 of human sequence)

Primer H2: 5'-GGATTAATACGGGACCATCCACACTACT (SEQ ID NO:41, based on NT2638-2611 of rat sequence)

The C1+C2 primer pair produced clones phG2-3-a and phGH2-3-b which havethe nucleic acid sequences of SEQ IDs 1 and 3 as their inserts,respectively. The D1+D2 primer pair produced phG2-4-a and phGH2-4-bwhich have the nucleic acid sequences of SEQ IDs 10 and 12 as theirinserts, respectively. The E1+E2 primer pair produced a clone which isbelieved to encompass nucleotides 1317-2379. The F1+F2 primer pairproduced a clone which is believed to encompass nucleotides 1447-2298.The G1+G2 primer pair produced clone phG2-7-a, which has the nucleicacid sequence of SEQ ID 14 as its insert and clone phG2-7-b which hasthe nucleic acid sequence of SEQ ID 16 as its insert. The H1+H2 primerpair produced phG2-8-a and phGH2-8-b which have the nucleic acidsequences of SEQ IDs 22 and 24 as their inserts, respectively.

The PCR fragments were cloned into pCR2.1 (Invitrogen). FIG. 1 shows thelocation of each of the cloned cDNAs in relation to the entire humanGlyT-2 sequence. Clone phG2-3 and phG2-8b were obtained from humancerebellum mRNA while the rest were from spinal cord. The cDNA insertswere sequenced using the AutoRead sequencing kit (Pharmacia) and theALFexpress™ automatic sequencing apparatus (Pharmacia). These sequencesimplied ten point variations in the amino acid sequence. The nucleicacid sequence of SEQ ID NO 18 is believed to represent the majorconsensus sequence. Comparison of the human GlyT-2 DNA sequence of SEQID NO 18 to the rat GlyT-2 sequence revealed an 89% nucleic acididentity and a 94.4% amino acid identity using the FASTA algorithm.

EXAMPLE 2

Full-length Clone

The human GlyT-2 cDNAs were then used to construct a full length humanGlyT-2 coding sequence, which was cloned into the pcDNA3 vector(Invitrogen). The clone incorporated the nucleic acid sequence of SEQ ID20 and was denoted pHGT2. The 5' end of the cDNA was constructed byinserting the 254 bp Hind III-Nar I fragment from clone phG2-3 intoclone phG2-1, previously digested with Hind III and Nar I. The 3' end ofthe cDNA was constructed be inserting the Hind III-Hinc II fragment fromphG2-2 and the Hinc II-Xba I fragment from clone phG2-7 into the pcDNA3vector previously digested with Hind IIII and Xba I. Lastly, the HindIII-Nru I fragment from the 5' end clone and the Nru I-Xba I fragmentfrom the 3' end clone were cloned into the pcDNA3 vector (Invitrogen)digested with Hind IIII and Xba I. The pHGT2 expression clone thusobtained contains the sequence of human GlyT-2 from 1 to 2397 under thecontrol of the human cytomegalovirus (CMV) promoter. In this expressionclone, nts 1-173 were derived from clone phG2-3; nts 174-823 werederived from clone phG2-1; nts 824-1599 were derived from clone phG2-2;and nts 1600-2397 were derived from clone phG2-7 (see FIG. 2).

EXAMPLE 3

Second Full-Length Clone

An expression clone containing the nucleic acid sequence of SEQ ID 18 isconstructed from the expression clone containing SEQ ID 20 bysite-directed mutagenesis to change NT 304 from G to A, NT 371 from T toC, NT 836 from A to T, NT 1116 from G to A, NT 1831 from G to A, NT 2382from T to C, NT 2388 from A to G, NT 2391 from T to C and NT 2394 from Ato G. The mutagenesis is conducted by the oligonucleotide-directedmethodology described by Ausubel et al, Current Protocols in MolecularBiology, John Wiley and Sons, New York, 1995, pp.8.1.1-8.1.6.

EXAMPLE 4

GlyT-2 Expression

The clone of example 2 was transfected into QT-6 cells (from AmericanType Culture Collection, Accession No. ATCC CRL-1708) using the methoddescribed in Example 5. The glycine transport assay described in Example6 was used to confirm that glycine transport activity was conferred tothe cells by the transfection.

EXAMPLE 5

Transfection

This example sets forth methods and materials used for growing andtransfecting QT-6 cells, which are avian fibroblasts derived from quail.Transfections with pHGT2 have been conducted, as have transfections withGlyT-1 vectors, though these latter transfections were conducted atseparate times.

QT-6 cells were obtained from American Type Culture Collection(Accession No. ATCC CRL-1708). Complete QT-6 medium for growing QT-6 wasMedium 199 (Sigma Chemical Company, St. Louis, Mo.; hereinafter "Sigma")supplemented to be 10% tryptose phosphate; 5% fetal bovine serum(Sigma); 1% penicillin-streptomycin (Sigma); and 1% steriledimethylsulfoxide (DMSO; Sigma). Other solutions required for growing ortransfecting QT-6 cells included:

DNA/DEAE Mix: 450 μl TBS, 450 μl DEAE Dextran (Sigma), and 100 μl of DNA(4 μg) in TE, where the DNA included GlyT-1a, GlyT-1b, GlyT-1c, orGlyT-2 encoding DNA, in a suitable expression vector. The DNA used wasas defined below.

PBS: Standard phosphate buffered saline, pH 7.4 including 1 mM CaCl₂ and1 mM MgCl₂ sterilized through a 0.2 μm filter.

TBS: One ml of Solution B, 10 ml of Solution A; brought to 100 ml withdistilled H₂ O; filter-sterilized and stored at 4° C.

TE: 0.01M Tris, 0.001M EDTA, pH 8.0.

DEAE dextran: Sigma, #D-9885. A stock solution was prepared consistingof 0.1% (1 mg/ml) of the DEAE dextran in TBS. The stock solution wasfilter sterilized and frozen in 1 ml aliquots.

Chloroquine: Sigma, #C-6628. A stock solution was prepared consisting of100 mM chloroquine in H₂ O. The stock solution was filter-sterilized andstored in 0.5 ml aliquots, frozen.

    ______________________________________           Solution A (10X):    ______________________________________           NaCl            8.00 g           KCl             0.38 g           Na.sub.2 HPO.sub.4                           0.20 g           Tris base       3.00 g    ______________________________________

The solution was adjusted to pH 7.5 with HCl, brought to 100.0 ml withdistilled H₂ O, and filter-sterilized and stored at room temperature.

    ______________________________________           Solution B (100X):    ______________________________________           CaCl.sub.2.2H.sub.2 O                            1.5 g           MgCl.sub.2.6H.sub.2 O                            1.0 g    ______________________________________

The solution was brought to 100 ml with distilled H₂ O, andfilter-sterilized; the solution was then stored at room temperature.

HBSS: 150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 10 mM glucose, 5 mM KCl, 1mM MgCl₂.H₂ O; adjusted with NaOH to pH 7.4.

Standard growth and passaging procedures used were as follows: Cellswere grown in 225 ml flasks. For passaging, cells were washed twice withwarm HBSS (5 ml each wash). Two ml of a 0.05% trypsin/EDTA solution wasadded, the culture was swirled, then the trypsin/EDTA solution wasaspirated quickly. The culture was then incubated about 2 minutes (untilcells lift off), then 10 ml of QT-6 media was added and the cells arefurther dislodged by swirling the flask and tapping its bottom. Thecells were removed and transferred to a 15 ml conical tube, centrifugedat 1000×g for 10 minutes, and resuspended in 10 ml of QT-6 medium. Asample was removed for counting, the cells were then diluted further toa concentration of 1×10⁵ cells/ml using QT-6 medium, and 65 ml of theculture was added per 225 ml flask of passaged cells.

Transfection was accomplished using cDNAs prepared as follows:

For human GlyT-2 expression, the pHGT2 clone described above was used.

The human GlyT-1a (hGlyT-1a) clone contained the sequence of hGlyT-1afrom nucleotide position 183 to 2108 cloned into the pRc/CMV vector(Invitrogen, San Diego, Calif.) as a Hind III-Xba I fragment asdescribed in Kim et al., Mol. Pharmacol., 45: 608-617, 1994. The first17 nucleotides (corresponding to the first 6 amino acids) of the GlyT-1asequence reported in this Kim et al. article is actually based on therat sequence. To determine whether the sequence of human GlyT-1a isdifferent in this region, the 5' region of hGlyT-1a from nucleotide 1 to212 was obtained by rapid amplification of cDNA ends using the 5' RACEsystem supplied by Gibco BRL (Gaithersburg, Md.). Sequencing of this 5'region of GlyT-1a confirmed that the first 17 nucleotides of codingsequence are identical in human and rat GlyT-1a.

The human GlyT-1b (hGlyT-1b) clone contained the sequence of hGlyT-1bfrom nucleotide position 213 to 2274 cloned into the pRc/CMV vector as aHind IIII-Xba I fragment as described in Kim et al., supra.

The human GlyT-1c (hGlyT-1c) clone contained the sequence of hGlyT-1cfrom nucleotide position 213 to 2336 cloned into the pRc/CMV vector(Invitrogen) as a Hind IIII-Xba I fragment as described in Kim et al.,supra. The Hind IIII-Xba fragment of hGlyT-1c from this clone wassubcloned into the pRc/RSV vector. Transfection experiments wereperformed with GlyT-1c in both the pRc/RSV and pRc/CMV expressionvectors.

The following four day procedure for the tranfections was used:

On day 1, QT-6 cells were plated at a density of 1×10⁶ cells in 10 ml ofcomplete QT-6 medium in 100 mm dishes.

On day 2, the medium was aspirated and the cells were washed with 10 mlof PBS followed by 10 ml of TBS. The TBS was aspirated, then 1 ml of theDEAE/DNA mix was added to the plate. The plate was swirled in the hoodevery 5 minutes. After 30 minutes, 8 ml of 80 μM chloroquine in QT-6medium was added and the culture was incubated for 2.5 hours at 37° C.and 5% CO₂. The medium was then aspirated and the cells were washed twotimes with complete QT-6 medium, then 100 ml complete QT-6 medium wasadded and the cells were returned to the incubator.

On day 3, the cells were removed with trypsin/EDTA as described above,and plated into the wells of 96-well assay plates at approximately 2×10⁵cells/well.

On day 4, glycine transport was assayed as described in Example 6.

EXAMPLE 6

Glycine Uptake

This example illustrates a method for the measurement of glycine uptakeby transfected cultured cells.

Transient GlyT-transfected cells or control cells grown in accordancewith Example 5 were washed three times with HEPES buffered saline (HBS).The control cells were treated precisely as the GlyT-transfected cellsexcept that the transfection procedure omitted any cDNA. The cells wereincubated 10 minutes at 37° C., after which a solution was addedcontaining 50 nM ³ H! glycine (17.5 Ci/mmol) and either (a) no potentialcompetitor, (b) 10 mM nonradioactive glycine or (c) a concentration of aprospective agent. A range of concentrations of the prospective agentwas used to generate data for calculating the concentration resulting in50% of the effect (for example, the IC₅₀ s, which are the concentrationsof agent inhibiting glycine uptake by 50%). The cells were thenincubated another 20 minutes at 37° C., after which the cells werewashed three times with ice-cold HBS. Scintillant was added to thecells, the cells were shaken for 30 minutes, and the radioactivity inthe cells was counted using a scintillation counter. Data were comparedbetween the cells contacted or not contacted by a prospective agent,and, where relevant, between cells having GlyT-1 activity versus cellshaving GlyT-2 activity, depending on the assay being conducted.

Expression of glycine transporter activity in QT-6 cells transfectedwith the human GlyT-2 clone, pHGT2, is demonstrated in FIG. 5, in which³ H! glycine uptake is shown for mock and pHGT2 transfected cells. QT-6cells transfected with pHGT2 show significant increases in glycinetransport as compared to mock transfected control cells. The results arepresented as means±SEM of a representative experiment performed intriplicate.

The concentration dependence of glycine transport in pHGT2-transfectedcells is shown in FIG. 6. QT-6 cells transfected with the human GlyT-2were incubated with 50 nM ³ H! glycine and the indicated concentrationsof unlabeled glycine for 20 minutes, and the cell-incorporatedradioactivity was determined by scintillation counting. Data pointsrepresent means±SEM from an experiment performed in quadruplicate. Theresults indicated an IC₅₀ of 40 μM.

EXAMPLE 7

Calcium Flux

This example illustrates a protocol for measuring calcium flux in cells.

The calcium flux measurement was generally performed in primary cellcultures, which were prepared using standard procedures and techniquesthat require sterile dissecting equipment, a microscope and definedmedium. The protocol used was substantially as described by Lu et al.,Proc. Nat'l. Acad. Sci. USA, 88: 6289-6292, 1991.

EXAMPLE 8

Binding to Strychnine-Sensitive Receptor

Binding of strychnine to strychnine-sensitive receptors was measured asdescribed in White et al. J. Neurochem. 35: 503-512, 1989 and Becker etal., J. Neurosci. 6: 1358-1364, 1986, with minor modifications.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 41    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 190 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #GGAGGCGGCG    60CTCCCAA GGAAATGAAT AAACTGCCAG CCAACAGCCC    #GCAGGAGCTT   120ACCCGGA TGGCCCATGC GCTCCCAGGA CGAGCCCGGA    #CGGCGCCCAA   180CCCCGCC GCCGCCACGT GTGCCCAGGT CCGCTTCCAC    #       190    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 63 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Lys Leu Pro Ala Asn Serro Lys Glu Met Asn    #                 15    #Asp Gly Pro Cys Ala Prola Gln Gly His Pro    #             30    #Ala Ala Ala Pro Pro Proln Glu Leu Pro Ala    #         45    #Ala Gln Thr Phe Glnrg Ser Ala Ser Thr Gly    #     60    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 190 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #GGAGGCGGCG    60CTCCCAA GGAAATGAAT AAACTGCCAG CCAACAGCCC    #GCAGGAGCTT   120ACCCGGA TGGCCCATGC GCTCCCAGGA CGAGCCCGGA    #CGGCGCCCAA   180CCCCGCC GCCGCCACGT GTGCCCAGGT CCGCTTCCAC    #       190    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 63 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #Lys Leu Pro Ala Asn Serro Lys Glu Met Asn    #                 15    #Asp Gly Pro Cys Ala Prola Gln Gly His Pro    #             30    #Ala Ala Ala Pro Pro Proln Glu Leu Pro Ala    #         45    #Ala Gln Thr Phe Glnrg Ser Ala Ser Thr Gly    #     60    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1216 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #GCGCTCCCAG    60GAGGCGG CGGCGGCGCA GGGCCACCCG GATGGCCCAT    #GTGTGCCCAG   120CAGGAGC TTCCCGCGGC TGCCGCCCCG CCGCCGCCAC    #GCGAGGCTGA   180GGCGCCC AAACTTTCCA GTCAGCGGAC GCGCGAGCCT    #CCTCTGCAGC   240GGGTCTT GCAAACTCAG TAGCCCGCGG GCGCAGGCGG    #GGAGCTCCGG   300AGAGAGG CGCAAGGCGC GCAGGCCTCG CCCCCTCCCG    #GGGATGCGAA   360CTGCACT GTAAGATCCC TTTTCTGCGA GGCCCGGAGG    #GCTGGGTGAA   420AAGGGCA CCCTGGAGCG GAACAATACC CCTGTTGTGG    #TCCCGGGCAG   480ACCGTGG TGCTGGGCAC GGATGGAATC ACGTCCGTGC    #CCCGAGGGAA   540GCCACCC AGGAGGACGA GCAAGGGGAT GAGAATAAGG    #GGCTGGGCAA   600CTGGACT TCATCCTGTC CATGGTGGGG TACGCAGTGG    #TCATCCCTTA   660CCCTACC TGGCCTTCCA GAACGGGGGA GGTGCTTTCC    #CGCTGGGCCA   720GCTCTGG CTGGATTACC CATCTTCTTC TTGGAGGTGT    #AAGGCTGTGG   780GGACCAG TGTCTGTGTG GAAGGCCATC CCAGCTCTAC    #TTATTTGCTA   840ATCATCT CTGTCCTAAT AGCCATATAC TACAATGTGA    #CCTGCAACAA   900CTGTTTG CCTCCTTTGT GTCTGTACTA CCCTGGGGCT    #CCTGTGTTAT   960CCAGAAT GCAAAGATAA AACCAAACTT TTATTAGATT    #CTTATCCCAA  1020AAAATAC AGATCAAGAA CTCGACTTTC TGCATGACCG    #GAAGTGAAGA  1080AATTTCA CCAGCCAGGC CAATAAGACA TTTGTCAGTG    #GCGAGATCGG  1140TTTGTGC TGAAGATTTC TGCAGGGATT GAATATCCTG    #CGTTGGCTAA  1200CTCTGCC TCTTCCTGGC TTGGGTCATT GTGTATGCAT    #  1216            A    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 405 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #Gly His Pro Asp Gly Prola Ala Ala Ala Gln    #                 15    #Leu Pro Ala Ala Ala Alaer Pro Glu Gln Glu    #             30    #Ser Thr Gly Ala Gln Thral Pro Arg Ser Ala    #         45    #Ala Glu Arg Pro Gly Valla Arg Ala Cys Glu    #     60    #Gln Ala Ala Ser Ala Alaer Ser Pro Arg Ala    # 80    #Gln Ala Ser Pro Pro Prolu Ala Gln Gly Ala    #                 95    #Cys Lys Ile Pro Phe Leuly Asn Ala Leu His    #            110    #Val Gly Lys Gly Thr Leusp Ala Asn Val Ser    #        125    #Val Asn Met Ser Gln Serro Val Val Gly Trp    #    140    #Ser Val Leu Pro Gly Serhr Asp Gly Ile Thr    #160    #Gln Gly Asp Glu Asn Lyshr Gln Glu Asp Glu    #                175    #Phe Ile Leu Ser Met Valer Ser Lys Leu Asp    #            190    #Arg Phe Pro Tyr Leu Alaeu Gly Asn Val Trp    #        205    #Pro Tyr Leu Met Met Leuly Ala Phe Leu Ile    #    220    #Glu Val Ser Leu Gly Glnro Ile Phe Phe Leu    #240    #Lys Ala Ile Pro Ala Leuro Val Ser Val Trp    #                255    #Ser Val Leu Ile Ala Ilela Met Leu Ile Ile    #            270    #Phe Tyr Leu Phe Ala Serle Cys Tyr Thr Leu    #        285    #Asn Asn Pro Trp Asn Thrro Trp Gly Ser Cys    #    300    #Leu Asp Ser Cys Val Ileys Thr Lys Leu Leu    #320    #Ser Thr Phe Cys Met Thrle Gln Ile Lys Asn    #                335    #Thr Ser Gln Ala Asn Lyshr Met Val Asn Phe    #            350    #Lys Tyr Phe Val Leu Lyser Glu Glu Tyr Phe    #        365    #Ile Gly Trp Pro Leu Alalu Tyr Pro Gly Glu    #    380    #Tyr Ala Ser Leu Ala Lysla Trp Val Ile Val    #400    -  Gly Ile Lys Thr Ser                     405    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1597 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #GCGCTCCCAG    60GAGGCGG CGGCGGCGCA GGGCCACCCG GATGGCCCAT    #GTGTGCCCAG   120CAGGAGC TTCCCGCGGC TGCCGCCCCG CCGCCGCCAC    #GCGAGGCTGA   180GGCGCCC AAACTTTCCA GTCAGCGGAC GCGCGAGCCT    #CCTCTGCAGC   240GGGTCTT GCAAACTCAG TAGCCCGCGG GCGCAGGCGG    #GGAGCTCCGG   300AGAGAGG CGCAAAGCGC GCAGGCCTCG CCCCCTCCCG    #GGGATGCGAA   360CTGCACT GTAAGATCCC TTCTCTGCGA GGCCCGGAGG    #GCTGGGTGAA   420AAGGGCA CCCTGGAGCG GAACAATACC CCTGTTGTGG    #TCCCGGGCAG   480ACCGTGG TGCTGGGCAC GGATGGAATC ACGTCCGTGC    #CCTGAGGGAA   540GCCACCC AGGAGGACGA GCAAGGGGAT GAGAATAAGG    #GGCTGGGCAA   600CTGGACT TCATCCTGTC CATGGTGGGG TACGCAGTGG    #TCATCCCTTA   660CCCTACC TGGCCTTCCA GAACGGGGGA GGTGCTTTCC    #CGCTGGGCCA   720GCTCTGG CTGGATTACC CATCTTCTTC TTGGAGGTGT    #AAGGCTGTGG   780GGACCAG TGTCTGTGTG GAAGGCCATC CCAGCTCTAC    #TTATTTGCTA   840ATCAACT CTGTCCTAAT AGCCATATAC TACAATGTGA    #CCTGCAACAA   900CTGTTTG CCTCCTTTGT GTCTGTACTA CCCTGGGGCT    #CCTGTGTTAT   960CCAGAAT GCAAAGATAA AACCAAACTT TTATTAGATT    #CTTATCCCAA  1020AAAATAC AGATCAAGAA CTCGACTTTC TGCATGACCG    #GAAGTGAGGA  1080AATTTCA CCAGCCAGGC CAATAAGACA TTTGTCAGTG    #GCGAGATCAG  1140TTTGTGC TGAAGATTTC TGCAGGGATT GAATATCCTG    #CGTTGGCTAA  1200CTCTGCC TCTTCCTGGC TTGGGTCATT GTGTATGCAT    #ATGTCGTACT  1260TCAGGAA AAGTGGTGTA CTTCACGGCC ACGTTCCCGT    #TCTGGTACTT  1320ATCCGAG GAGTCACCCT GCCTGGAGCT GGAGCTGGGA    #CTGCCACTCA  1380TGGGAGA AACTCACGGA TGCCACGGTG TGGAAAGATG    #CTTACAACAA  1440TTATCTG CTGCATGGGG AGGCCTGATC ACTCTCTCTT    #GTGCCACAAG  1500TGCTACA GGGACACTCT AATTGTCACC TGCACCAACA    #AACGCAAAGT  1560TTCGTCA TCTTCTCCGT TATCGGCTTC ATGGCCAATG    #    1597          GGCAG ACCAAGGGCC AGGCATT    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 177 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #Gly His Pro Asp Gly Prola Ala Ala Ala Gln    #                 15    #Leu Pro Ala Ala Ala Alaer Pro Glu Gln Glu    #             30    #Ser Thr Gly Ala Gln Thral Pro Arg Ser Ala    #         45    #Ala Glu Arg Pro Gly Valla Arg Ala Cys Glu    #     60    #Gln Ala Ala Ser Ala Alaer Ser Pro Arg Ala    # 80    #Gln Ala Ser Pro Pro Prolu Ala Gln Ser Ala    #                 95    #Cys Lys Ile Pro Ser Leuly Asn Ala Leu His    #            110    #Val Gly Lys Gly Thr Leusp Ala Asn Val Ser    #        125    #Val Asn Met Ser Gln Serro Val Val Gly Trp    #    140    #Ser Val Leu Pro Gly Serhr Asp Gly Ile Thr    #160    #Gln Gly Asp Glu Asn Lyshr Gln Glu Asp Glu    #                175    -  Ala    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 354 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #Leu Ser Met Val Gly Tyrys Leu Asp Phe Ile    #                 15    #Pro Tyr Leu Ala Phe Glnsn Val Trp Arg Phe    #             30    #Leu Met Met Leu Ala Leuhe Leu Ile Pro Tyr    #         45    #Ser Leu Gly Gln Phe Alahe Phe Leu Glu Val    #     60    #Ile Pro Ala Leu Gln Glyer Val Trp Lys Ala    # 80    #Leu Ile Ala Ile Tyr Tyreu Ile Asn Ser Val    #                 95    #Leu Phe Ala Ser Phe Valyr Thr Leu Phe Tyr    #            110    #Pro Trp Asn Thr Pro Gluly Ser Cys Asn Asn    #        125    #Ser Cys Val Ile Ser Aspys Leu Leu Leu Asp    #    140    #Phe Cys Met Thr Ala Tyrle Lys Asn Ser Thr    #160    #Gln Ala Asn Lys Thr Pheal Asn Phe Thr Ser    #                175    #Phe Val Leu Lys Ile Serlu Tyr Phe Lys Tyr    #            190    #Trp Pro Leu Ala Leu Cysro Gly Glu Ile Arg    #        205    #Ser Leu Ala Lys Gly Ileal Ile Val Tyr Ala    #    220    #Ala Thr Phe Pro Tyr Valal Val Tyr Phe Thr    #240    #Thr Leu Pro Gly Ala Glyeu Ile Arg Gly Val    #                255    #Trp Glu Lys Leu Thr Asphe Ile Thr Pro Lys    #            270    #Ile Phe Phe Ser Leu Sersp Ala Ala Thr Gln    #        285    #Ser Tyr Asn Lys Phe Hiseu Ile Thr Leu Ser    #    300    #Thr Cys Thr Asn Ser Alasp Thr Leu Ile Val    #320    #Ser Val Ile Gly Phe Metly Phe Val Ile Phe    #                335    #Val Ala Asp Gln Gly Proal Asn Ile Glu Asn    #            350    -  Gly Ile    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 533 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #CCAACTGGGA    60CCTGGAG CTGGAGCTGG GATCTGGTAC TTCATCACAC    #TCTCTTTATC   120GCCACGG TGTGGAAAGA TGCTGCCACT CAGATTTTCT    #ACAACTGCTA   180GGCCTGA TCACTCTCTC TTCTTACAAC AAATTCCACA    #CCGGCTTCGT   240ATTGTCA CCTGCACCAA CAGTGCCACA AGCATCTTTG    #AGAATGTGGC   300ATCGGCT TCATGGCCAA TGAACGCAAA GTCAACATTG    #GGCTGCCTCT   360GGCATTG CATTTGTGGT TTACCCGGAA GCCTTAACCA    #TTGACACTAT   420GCCATCA TCTTTTTCCT GATGCTCCTC ACTCTTGGAC    #AGTACCTACG   480GAGACCA TAGTGACCTC CATCTCAGAC GAGTTTCCCA    #TGG          533GTGTTTA CTCTGGGCTG CTGCATTTGT TTCTTCATCA    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 177 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #Ile Trp Tyr Phe Ile Thrly Ala Gly Ala Gly    #                 15    #Val Trp Lys Asp Ala Alaeu Thr Asp Ala Thr    #             30    #Trp Gly Gly Leu Ile Threr Leu Ser Ala Ala    #         45    #Cys Tyr Arg Asp Thr Leuys Phe His Asn Asn    #     60    #Ile Phe Ala Gly Phe Valsn Ser Ala Thr Ser    # 80    #Glu Arg Lys Val Asn Ilely Phe Met Ala Asn    #                 95    #Ala Phe Val Val Tyr Proln Gly Pro Gly Ile    #            110    #Phe Trp Ala Ile Ile Pheeu Pro Leu Ser Pro    #        125    #Thr Met Phe Ala Thr Ilehr Leu Gly Leu Asp    #    140    #Phe Pro Lys Tyr Leu Arger Ile Ser Asp Glu    #160    #Cys Ile Cys Phe Phe Ilehe Thr Leu Gly Cys    #                175    -  Met    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 533 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #CCAAGTGGGA    60CCTGGAG CTGGAGCTGG GATCTGGTAC TTCATCACAC    #TCTCTTTATC   120GCCACGG TGTGGAAAGA TGCTGCCACT CAGATTTTCT    #ACAACTGCTA   180GGCCTGA TCACTCTCTC TTCTTACAAC AAATTCCACA    #CCGGCTTCGT   240ATTGTCA CCTGCACCAA CAGTGCCACA AGCATCTTTG    #AGAATGTGGC   300ATCGGCT TCATGGCCAA TGAACGCAAA GTCAACATTG    #GGCTGCCTCT   360GGCATTG CATTTGTGGT TTACCCGGAA GCCTTAACCA    #TTGACACTAT   420GCCATCA TCTTTTTCCT GATGCTCCTC ACTCTTGGAC    #AGTACCTACG   480GAGACCA TAGTGACCTC CATCTCAGAC GAGTTTCCCA    #TGG          533GTGTTTA CTCTGGGCTG CTGCATTTGT TTCTTCATCA    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 177 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #Ile Trp Tyr Phe Ile Thrly Ala Gly Ala Gly    #                 15    #Val Trp Lys Asp Ala Alaeu Thr Asn Ala Thr    #             30    #Trp Gly Gly Leu Ile Threr Leu Ser Ala Ala    #         45    #Cys Tyr Arg Asp Thr Leuys Phe His Asn Asn    #     60    #Ile Phe Ala Gly Phe Valsn Ser Ala Thr Ser    # 80    #Glu Arg Lys Val Asn Ilely Phe Met Ala Asn    #                 95    #Ala Phe Val Val Tyr Proln Gly Pro Gly Ile    #            110    #Phe Trp Ala Ile Ile Pheeu Pro Leu Ser Pro    #        125    #Thr Met Phe Ala Thr Ilehr Leu Gly Leu Asp    #    140    #Phe Pro Lys Tyr Leu Arger Ile Ser Asp Glu    #160    #Cys Ile Cys Phe Phe Ilehe Thr Leu Gly Cys    #                175    -  Met    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 840 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #ACGCAAAGTC    60TCGTCAT CTTCTCCGTT ATCGGCTTCA TGGCCAATGA    #CCCGGAAGCC   120TGGCAGA CCAAGGGCCA GGCATTGCAT TTGTGGTTTA    #GCTCCTCACT   180CTCTCTC TCCGTTCTGG GCCATCATCT TTTTCCTGAT    #CTCAGACGAG   240CTATGTT TGCCACCATC GAGACCATAG TGACCTCCAT    #CGTTTGTTTC   300TACGCAC ACACAAGCCA GTGTTTACTC TGGGCTGCTG    #GCTTGTGGAC   360TTCCAAT GATCACTCAG GGTGGAATTT ACATGTTTCA    #GGGGATCTCT   420CCTATGC CCTTGTCATC ATTGCCATTT TTGAGCTCGT    #ATTCCAGCCT   480TGCAAAG ATTCTGTGAA GATATAGAGA TGATGATTGG    #CTTTATCCTT   540AAGTCTG CTGGGCATTT GTAACCCCAA CCATTTTAAC    #TCCTAACTGG   600ACCAGTG GGAGCCCATG ACCTATGGCT CTTACCGCTA    #AATTATGTTT   660GATGGCT AATGCTCGCC TGTTCCGTCA TCTGGATCCC    #GGTGTGCTCG   720ATCTGGC CCCTGGAAGA TTTATTGAGA GGCTGAAGTT    #CAAGAACATG   780GGGGCCC ATTCTTAGCT CAACACCGCG GGGAGCGTTA    #TTTGGAACTG   840GAACCTC TTCCTTGGGA CTCAAACTGC CAGTGAAGGA    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 280 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #Ile Gly Phe Met Ala Asnal Ile Phe Ser Val    #                 15    #Asp Gln Gly Pro Gly Ilele Glu Asn Val Ala    #             30    #Arg Leu Pro Leu Ser Proro Glu Ala Leu Thr    #         45    #Leu Thr Leu Gly Leu Asphe Phe Leu Met Leu    #     60    #Thr Ser Ile Ser Asp Glule Glu Thr Ile Val    # 80    #Val Phe Thr Leu Gly Cysrg Thr His Lys Pro    #                 95    #Met Ile Thr Gln Gly Glyle Met Gly Phe Pro    #            110    #Ala Ala Ser Tyr Ala Leueu Val Asp Thr Tyr    #        125    #Ile Ser Tyr Val Tyr Glyhe Glu Leu Val Gly    #    140    #Met Ile Gly Phe Gln Prolu Asp Ile Glu Met    #160    #Val Thr Pro Thr Ile Leual Cys Trp Ala Phe    #                175    #Trp Glu Pro Met Thr Tyrhe Ser Phe Tyr Gln    #            190    #Val Leu Gly Trp Leu Metro Asn Trp Ser Met    #        205    #Met Phe Val Ile Lys Metle Trp Ile Pro Ile    #    220    #Leu Lys Leu Val Cys Serrg Phe Ile Glu Arg    #240    #Gln His Arg Gly Glu Argly Pro Phe Leu Ala    #                255    #Ser Ser Leu Gly Leu Lyssp Pro Leu Gly Thr    #            270    -  Leu Pro Val Lys Asp Leu Glu Leu    #        280    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 840 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #ACGCAAAGTC    60TCGTCAT CTTCTCCGTT ATCGGCTTCA TGGCCAATGA    #CCCGGAAGCC   120TGGCAGA CCAAGGGCCA GGCATTGCAT TTGTGGTTTA    #GCTCCTCACT   180CTCTCTC TCCGTTCTGG GCCATCATCT TTTTCCTGAT    #CTCAGACGAG   240CTATGTT TGCCACCATC GAGACCATAG TGACCTCCAT    #CATTTGTTTC   300TACGCAC ACACAAGCCA GTGTTTACTC TGGGCTGCTG    #GCTTGTGGAC   360TTCCAAT GATCACTCAG GGTGGAATTT ACATGTTTCA    #GGGGATCTCT   420CCTATGC CCTTGTCATC ATTGCCATTT TTGAGCTCGT    #ATTCCAGCCT   480TGCAAAG ATTCTGTGAA GATATAGAGA TGATGATTGG    #CTTTATCCTT   540AAGTCTG CTGGGCATTT GTAACCCCAA CCATTTTAAC    #TCCTAACTGG   600ACCAGTG GGAGCCCATG ACCTATGGCT CTTACCGCTA    #AATTATGTTT   660GATGGCT AATGCTCGCC TGTTCCGTCA TCTGGATCCC    #GGTGTGCTCG   720ATCTGGC CCCTGGAAGA TTTATTGAGA GGCTGAAGTT    #CAAGAACATG   780GGGGCCC ATTCTTAGCT CAACACCGCG GGGAGCGTTA    #TTTGGAACTG   840GAACCTC TTCCTTGGGA CTCAAACTGC CAGTGAAGGA    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 280 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #Ile Gly Phe Met Ala Asnal Ile Phe Ser Val    #                 15    #Asp Gln Gly Pro Gly Ilele Glu Asn Val Ala    #             30    #Arg Leu Pro Leu Ser Proro Glu Ala Leu Thr    #         45    #Leu Thr Leu Gly Leu Asphe Phe Leu Met Leu    #     60    #Thr Ser Ile Ser Asp Glule Glu Thr Ile Val    # 80    #Val Phe Thr Leu Gly Cysrg Thr His Lys Pro    #                 95    #Met Ile Thr Gln Gly Glyle Met Gly Phe Pro    #            110    #Ala Ala Ser Tyr Ala Leueu Val Asp Thr Tyr    #        125    #Ile Ser Tyr Val Tyr Glyhe Glu Leu Val Gly    #    140    #Met Ile Gly Phe Gln Prolu Asp Ile Glu Met    #160    #Val Thr Pro Thr Ile Leual Cys Trp Ala Phe    #                175    #Trp Glu Pro Met Thr Tyrhe Ser Phe Tyr Gln    #            190    #Val Leu Gly Trp Leu Metro Asn Trp Ser Met    #        205    #Met Phe Val Ile Lys Metle Trp Ile Pro Ile    #    220    #Leu Lys Leu Val Cys Serrg Phe Ile Glu Arg    #240    #Gln His Arg Gly Glu Argly Pro Phe Leu Ala    #                255    #Ser Ser Leu Gly Leu Lyssp Pro Leu Gly Thr    #            270    -  Leu Pro Val Lys Asp Leu Glu Leu    #        280    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2397 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #GGAGGCGGCG    60CTCCCAA GGAAATGAAT AAACTGCCAG CCAACAGCCC    #GCAGGAGCTT   120ACCCGGA TGGCCCATGC GCTCCCAGGA CGAGCCCGGA    #CGGCGCCCAA   180CCCCGCC GCCGCCACGT GTGCCCAGGT CCGCTTCCAC    #GGGGTCTTGC   240CGGACGC GCGAGCCTGC GAGGCTGAGC GGCCAGGAGT    #GAGAGAGGCG   300CGCGGGC GCAGGCGGCC TCTGCAGCTC TGCGGGACTT    #GCTGCACTGT   360CCTCGCC CCCTCCCGGG AGCTCCGGGC CCGGCAACGC    #CAAGGGCACC   420TGCGAGG CCCGGAGGGG GATGCGAACG TGAGTGTGGG    #CACCGTGGTG   480ATACCCC TGTTGTGGGC TGGGTGAACA TGAGCCAGAG    #TGCCACCCAG   540GAATCAC GTCCGTGCTC CCGGGCAGCG TGGCCACCGT    #ACTGGACTTC   600GGGATGA GAATAAGGCC CGAGGGAACT GGTCCAGCAA    #TCCCTACCTG   660TGGGGTA CGCAGTGGGG CTGGGCAATG TCTGGAGGTT    #GGCTCTGGCT   720GGGGAGG TGCTTTCCTC ATCCCTTACC TGATGATGCT    #GGGACCAGTG   780TCTTCTT GGAGGTGTCG CTGGGCCAGT TTGCCAGCCA    #GATCATCTCT   840CCATCCC AGCTCTACAA GGCTGTGGCA TCGCGATGCT    #CCTGTTTGCC   900TATACTA CAATGTGATT ATTTGCTATA CACTTTTCTA    #GCCAGAATGC   960TACTACC CTGGGGCTCC TGCAACAACC CTTGGAATAC    #CAAAATACAG  1020AACTTTT ATTAGATTCC TGTGTTATCA GTGACCATCC    #TAATTTCACC  1080CTTTCTG CATGACCGCT TATCCCAACG TGACAATGGT    #CTTTGTGCTG  1140AGACATT TGTCAGTGGA AGTGAAGAGT ACTTCAAGTA    #TCTCTGCCTC  1200GGATTGA ATATCCTGGC GAGATCAGGT GGCCACTAGC    #TTCAGGAAAA  1260TCATTGT GTATGCATCG TTGGCTAAAG GAATCAAGAC    #CATCCGAGGA  1320CGGCCAC GTTCCCGTAT GTCGTACTCG TGATCCTCCT    #GTGGGAGAAA  1380GAGCTGG AGCTGGGATC TGGTACTTCA TCACACCCAA    #TTTATCTGCT  1440CGGTGTG GAAAGATGCT GCCACTCAGA TTTTCTTCTC    #CTGCTACAGG  1500TGATCAC TCTCTCTTCT TACAACAAAT TCCACAACAA    #CTTCGTCATC  1560TCACCTG CACCAACAGT GCCACAAGCA TCTTTGCCGG    #TGTGGCAGAC  1620GCTTCAT GGCCAATGAA CGCAAAGTCA ACATTGAGAA    #GCCTCTCTCT  1680TTGCATT TGTGGTTTAC CCGGAAGCCT TAACCAGGCT    #CACTATGTTT  1740TCATCTT TTTCCTGATG CTCCTCACTC TTGGACTTGA    #CCTACGCACA  1800CCATAGT GACCTCCATC TCAGACGAGT TTCCCAAGTA    #TTTTCCAATG  1860TTACTCT GGGCTGCTGC ATTTGTTTCT TCATCATGGG    #CTCCTATGCC  1920GAATTTA CATGTTTCAG CTTGTGGACA CCTATGCTGC    #CTTGCAAAGA  1980CCATTTT TGAGCTCGTG GGGATCTCTT ATGTGTATGG    #GAAAGTCTGC  2040TAGAGAT GATGATTGGA TTCCAGCCTA ACATCTTCTG    #TTACCAGTGG  2100CCCCAAC CATTTTAACC TTTATCCTTT GCTTCAGCTT    #CGGATGGCTA  2160ATGGCTC TTACCGCTAT CCTAACTGGT CCATGGTGCT    #GCATCTGGCC  2220CCGTCAT CTGGATCCCA ATTATGTTTG TGATAAAAAT    #CTGGGGCCCA  2280TTGAGAG GCTGAAGTTG GTGTGCTCGC CACAGCCGGA    #GGGAACCTCT  2340ACCGCGG GGAGCGTTAC AAGAACATGA TCGACCCCTT    #CTAGTCC     2397AACTGCC AGTGAAGGAT TTGGAACTGG GCACTCAGTG    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 797 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    #Lys Leu Pro Ala Asn Serro Lys Glu Met Asn    #                 15    #Asp Gly Pro Cys Ala Prola Gln Gly His Pro    #             30    #Ala Ala Ala Pro Pro Proln Glu Leu Pro Ala    #         45    #Ala Gln Thr Phe Gln Serer Ala Ser Thr Gly    #     60    #Pro Gly Val Gly Ser Cysys Glu Ala Glu Arg    # 80    #Ser Ala Ala Leu Arg Asprg Ala Gln Ala Ala    #                 95    #Pro Pro Pro Gly Ser Serer Ala Gln Ala Ser    #            110    #Pro Ser Leu Arg Gly Proeu His Cys Lys Ile    #        125    #Gly Thr Leu Glu Arg Asnal Ser Val Gly Lys    #    140    #Ser Gln Ser Thr Val Vally Trp Val Asn Met    #160    #Pro Gly Ser Val Ala Thrle Thr Ser Val Leu    #                175    #Glu Asn Lys Ala Arg Glysp Glu Gln Gly Asp    #            190    #Ser Met Val Gly Tyr Alaeu Asp Phe Ile Leu    #        205    #Tyr Leu Ala Phe Gln Asnal Trp Arg Phe Pro    #    220    #Met Met Leu Ala Leu Alaeu Ile Pro Tyr Leu    #240    #Leu Gly Gln Phe Ala Serhe Leu Glu Val Ser    #                255    #Pro Ala Leu Gln Gly Cysal Trp Lys Ala Ile    #            270    #Ile Ala Ile Tyr Tyr Asnle Ile Ser Val Leu    #        285    #Phe Ala Ser Phe Val Serhr Leu Phe Tyr Leu    #    300    #Trp Asn Thr Pro Glu Cyser Cys Asn Asn Pro    #320    #Cys Val Ile Ser Asp Hiseu Leu Leu Asp Ser    #                335    #Cys Met Thr Ala Tyr Proys Asn Ser Thr Phe    #            350    #Ala Asn Lys Thr Phe Valsn Phe Thr Ser Gln    #        365    #Val Leu Lys Ile Ser Alayr Phe Lys Tyr Phe    #    380    #Pro Leu Ala Leu Cys Leuly Glu Ile Arg Trp    #400    #Leu Ala Lys Gly Ile Lysle Val Tyr Ala Ser    #                415    #Thr Phe Pro Tyr Val Valal Tyr Phe Thr Ala    #            430    #Leu Pro Gly Ala Gly Alale Arg Gly Val Thr    #        445    #Glu Lys Leu Thr Asp Alale Thr Pro Lys Trp    #    460    #Phe Phe Ser Leu Ser Alala Ala Thr Gln Ile    #480    #Tyr Asn Lys Phe His Asnle Thr Leu Ser Ser    #                495    #Cys Thr Asn Ser Ala Thrhr Leu Ile Val Thr    #            510    #Val Ile Gly Phe Met Alahe Val Ile Phe Ser    #        525    #Ala Asp Gln Gly Pro Glysn Ile Glu Asn Val    #    540    #Thr Arg Leu Pro Leu Seryr Pro Glu Ala Leu    #560    #Leu Leu Thr Leu Gly Leule Phe Phe Leu Met    #                575    #Val Thr Ser Ile Ser Asphr Ile Glu Thr Ile    #            590    #Pro Val Phe Thr Leu Glyeu Arg Thr His Lys    #        605    #Pro Met Ile Thr Gln Glyhe Ile Met Gly Phe    #    620    #Tyr Ala Ala Ser Tyr Alaln Leu Val Asp Thr    #640    #Gly Ile Ser Tyr Val Tyrle Phe Glu Leu Val    #                655    #Met Met Ile Gly Phe Glnys Glu Asp Ile Glu    #            670    #Phe Val Thr Pro Thr Ileys Val Cys Trp Ala    #        685    #Gln Trp Glu Pro Met Thrys Phe Ser Phe Tyr    #    700    #Met Val Leu Gly Trp Leuyr Pro Asn Trp Ser    #720    #Ile Met Phe Val Ile Lysal Ile Trp Ile Pro    #                735    #Arg Leu Lys Leu Val Cysly Arg Phe Ile Glu    #            750    #Ala Gln His Arg Gly Glurp Gly Pro Phe Leu    #        765    #Thr Ser Ser Leu Gly Leule Asp Pro Leu Gly    #    780    #Thr Gln Cysro Val Lys Asp Leu Glu Leu Gly    #795    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2397 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    #GGAGGCGGCG    60CTCCCAA GGAAATGAAT AAACTGCCAG CCAACAGCCC    #GCAGGAGCTT   120ACCCGGA TGGCCCATGC GCTCCCAGGA CGAGCCCGGA    #CGGCGCCCAA   180CCCCGCC GCCGCCACGT GTGCCCAGGT CCGCTTCCAC    #GGGGTCTTGC   240CGGACGC GCGAGCCTGC GAGGCTGAGC GGCCAGGAGT    #GAGAGAGGCG   300CGCGGGC GCAGGCGGCC TCTGCAGCTC TGCGGGACTT    #GCTGCACTGT   360CCTCGCC CCCTCCCGGG AGCTCCGGGC CCGGCAACGC    #CAAGGGCACC   420TGCGAGG CCCGGAGGGG GATGCGAACG TGAGTGTGGG    #CACCGTGGTG   480ATACCCC TGTTGTGGGC TGGGTGAACA TGAGCCAGAG    #TGCCACCCAG   540GAATCAC GTCCGTGCTC CCGGGCAGCG TGGCCACCGT    #ACTGGACTTC   600GGGATGA GAATAAGGCC CGAGGGAACT GGTCCAGCAA    #TCCCTACCTG   660TGGGGTA CGCAGTGGGG CTGGGCAATG TCTGGAGGTT    #GGCTCTGGCT   720GGGGAGG TGCTTTCCTC ATCCCTTACC TGATGATGCT    #GGGACCAGTG   780TCTTCTT GGAGGTGTCG CTGGGCCAGT TTGCCAGCCA    #GATCAACTCT   840CCATCCC AGCTCTACAA GGCTGTGGCA TCGCGATGCT    #CCTGTTTGCC   900TATACTA CAATGTGATT ATTTGCTATA CACTTTTCTA    #GCCAGAATGC   960TACTACC CTGGGGCTCC TGCAACAACC CTTGGAATAC    #CAAAATACAG  1020AACTTTT ATTAGATTCC TGTGTTATCA GTGACCATCC    #TAATTTCACC  1080CTTTCTG CATGACCGCT TATCCCAACG TGACAATGGT    #CTTTGTGCTG  1140AGACATT TGTCAGTGGA AGTGAGGAGT ACTTCAAGTA    #TCTCTGCCTC  1200GGATTGA ATATCCTGGC GAGATCAGGT GGCCACTAGC    #TTCAGGAAAA  1260TCATTGT GTATGCATCG TTGGCTAAAG GAATCAAGAC    #CATCCGAGGA  1320CGGCCAC GTTCCCGTAT GTCGTACTCG TGATCCTCCT    #GTGGGAGAAA  1380GAGCTGG AGCTGGGATC TGGTACTTCA TCACACCCAA    #TTTATCTGCT  1440CGGTGTG GAAAGATGCT GCCACTCAGA TTTTCTTCTC    #CTGCTACAGG  1500TGATCAC TCTCTCTTCT TACAACAAAT TCCACAACAA    #CTTCGTCATC  1560TCACCTG CACCAACAGT GCCACAAGCA TCTTTGCCGG    #TGTGGCAGAC  1620GCTTCAT GGCCAATGAA CGCAAAGTCA ACATTGAGAA    #GCCTCTCTCT  1680TTGCATT TGTGGTTTAC CCGGAAGCCT TAACCAGGCT    #CACTATGTTT  1740TCATCTT TTTCCTGATG CTCCTCACTC TTGGACTTGA    #CCTACGCACA  1800CCATAGT GACCTCCATC TCAGACGAGT TTCCCAAGTA    #TTTTCCAATG  1860TTACTCT GGGCTGCTGC GTTTGTTTCT TCATCATGGG    #CTCCTATGCC  1920GAATTTA CATGTTTCAG CTTGTGGACA CCTATGCTGC    #CTTGCAAAGA  1980CCATTTT TGAGCTCGTG GGGATCTCTT ATGTGTATGG    #GAAAGTCTGC  2040TAGAGAT GATGATTGGA TTCCAGCCTA ACATCTTCTG    #TTACCAGTGG  2100CCCCAAC CATTTTAACC TTTATCCTTT GCTTCAGCTT    #CGGATGGCTA  2160ATGGCTC TTACCGCTAT CCTAACTGGT CCATGGTGCT    #GCATCTGGCC  2220CCGTCAT CTGGATCCCA ATTATGTTTG TGATAAAAAT    #CTGGGGCCCA  2280TTGAGAG GCTGAAGTTG GTGTGCTCGC CACAGCCGGA    #GGGAACCTCT  2340ACCGCGG GGAGCGTTAC AAGAACATGA TCGACCCCTT    #TTAATCC     2397AACTGCC AGTGAAGGAT TTGGAACTGG GTACTCAATG    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 797 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    #Lys Leu Pro Ala Asn Serro Lys Glu Met Asn    #                 15    #Asp Gly Pro Cys Ala Prola Gln Gly His Pro    #             30    #Ala Ala Ala Pro Pro Proln Glu Leu Pro Ala    #         45    #Ala Gln Thr Phe Gln Serer Ala Ser Thr Gly    #     60    #Pro Gly Val Gly Ser Cysys Glu Ala Glu Arg    # 80    #Ser Ala Ala Leu Arg Asprg Ala Gln Ala Ala    #                 95    #Pro Pro Pro Gly Ser Serly Ala Gln Ala Ser    #            110    #Pro Phe Leu Arg Gly Proeu His Cys Lys Ile    #        125    #Gly Thr Leu Glu Arg Asnal Ser Val Gly Lys    #    140    #Ser Gln Ser Thr Val Vally Trp Val Asn Met    #160    #Pro Gly Ser Val Ala Thrle Thr Ser Val Leu    #                175    #Glu Asn Lys Ala Arg Glysp Glu Gln Gly Asp    #            190    #Ser Met Val Gly Tyr Alaeu Asp Phe Ile Leu    #        205    #Tyr Leu Ala Phe Gln Asnal Trp Arg Phe Pro    #    220    #Met Met Leu Ala Leu Alaeu Ile Pro Tyr Leu    #240    #Leu Gly Gln Phe Ala Serhe Leu Glu Val Ser    #                255    #Pro Ala Leu Gln Gly Cysal Trp Lys Ala Ile    #            270    #Ile Ala Ile Tyr Tyr Asnle Asn Ser Val Leu    #        285    #Phe Ala Ser Phe Val Serhr Leu Phe Tyr Leu    #    300    #Trp Asn Thr Pro Glu Cyser Cys Asn Asn Pro    #320    #Cys Val Ile Ser Asp Hiseu Leu Leu Asp Ser    #                335    #Cys Met Thr Ala Tyr Proys Asn Ser Thr Phe    #            350    #Ala Asn Lys Thr Phe Valsn Phe Thr Ser Gln    #        365    #Val Leu Lys Ile Ser Alayr Phe Lys Tyr Phe    #    380    #Pro Leu Ala Leu Cys Leuly Glu Ile Arg Trp    #400    #Leu Ala Lys Gly Ile Lysle Val Tyr Ala Ser    #                415    #Thr Phe Pro Tyr Val Valal Tyr Phe Thr Ala    #            430    #Leu Pro Gly Ala Gly Alale Arg Gly Val Thr    #        445    #Glu Lys Leu Thr Asp Alale Thr Pro Lys Trp    #    460    #Phe Phe Ser Leu Ser Alala Ala Thr Gln Ile    #480    #Tyr Asn Lys Phe His Asnle Thr Leu Ser Ser    #                495    #Cys Thr Asn Ser Ala Thrhr Leu Ile Val Thr    #            510    #Val Ile Gly Phe Met Alahe Val Ile Phe Ser    #        525    #Ala Asp Gln Gly Pro Glysn Ile Glu Asn Val    #    540    #Thr Arg Leu Pro Leu Seryr Pro Glu Ala Leu    #560    #Leu Leu Thr Leu Gly Leule Phe Phe Leu Met    #                575    #Val Thr Ser Ile Ser Asphr Ile Glu Thr Ile    #            590    #Pro Val Phe Thr Leu Glyeu Arg Thr His Lys    #        605    #Pro Met Ile Thr Gln Glyhe Ile Met Gly Phe    #    620    #Tyr Ala Ala Ser Tyr Alaln Leu Val Asp Thr    #640    #Gly Ile Ser Tyr Val Tyrle Phe Glu Leu Val    #                655    #Met Met Ile Gly Phe Glnys Glu Asp Ile Glu    #            670    #Phe Val Thr Pro Thr Ileys Val Cys Trp Ala    #        685    #Gln Trp Glu Pro Met Thrys Phe Ser Phe Tyr    #    700    #Met Val Leu Gly Trp Leuyr Pro Asn Trp Ser    #720    #Ile Met Phe Val Ile Lysal Ile Trp Ile Pro    #                735    #Arg Leu Lys Leu Val Cysly Arg Phe Ile Glu    #            750    #Ala Gln His Arg Gly Glurp Gly Pro Phe Leu    #        765    #Thr Ser Ser Leu Gly Leule Asp Pro Leu Gly    #    780    #Thr Gln Cysro Val Lys Asp Leu Glu Leu Gly    #795    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 589 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    #TGATCACTCA    60GGCTGCT ACATTTGTTT CTTCATCATG GGTTTTCCAA    #CCCTTGTCAT   120ATGTTTC AGCTTGTGGA CACCTATGCT GCCTCCTATG    #GATTCTGTGA   180GAGCTCG TGGGGATCTC TTATGTGTAT GGCTTGCAAA    #GCTGGGCATT   240ATGATTG GATTCCAGCC TAACATCTTC TGGAAAGTCT    #GGGAGCCCAT   300ATTTTAA CCTTTATCCT TTGCTTCAGC TTTTACCAGT    #TAATGCTCGC   360TACCGCT ATCCTAACTG GTCCATGGTG CTCGGATGGC    #CCCCTGGAAG   420TGGATCC CAATTATGTT TGTGGTAAAA ATGCATCTGG    #CATTCTTAGC   480CTGAAGT TGGTGTGCTC GCCACAGCCG GACTGGGGCC    #CTTCCTTGGG   540GAGCGTT ACAAGAACAT GATCGACCCC TTGGGAACCT    #              589TGAAGG ATTTGGAACT GGGCACTCAG TGCTAGTCC    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 194 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    #Phe Ile Met Gly Phe Proys Tyr Ile Cys Phe    #                 15    #Gln Leu Val Asp Thr Tyrly Ile Tyr Met Phe    #             30    #Ile Phe Glu Leu Val Glyeu Val Ile Ile Ala    #         45    #Cys Glu Asp Ile Glu Metly Leu Gln Arg Phe    #     60    #Lys Val Cys Trp Ala Phero Asn Ile Phe Trp    # 80    #Cys Phe Ser Phe Tyr Glneu Thr Phe Ile Leu    #                 95    #Tyr Pro Asn Trp Ser Metyr Gly Ser Tyr Arg    #            110    #Val Ile Trp Ile Pro Ileet Leu Ala Cys Ser    #        125    #Gly Arg Phe Ile Glu Arget His Leu Ala Pro    #    140    #Trp Gly Pro Phe Leu Alaer Pro Gln Pro Asp    #160    #Ile Asp Pro Leu Gly Thrrg Tyr Lys Asn Met    #                175    #Asp Leu Glu Leu Gly Thrys Leu Pro Val Lys    #            190    -  Gln Cys    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 589 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    #TGATCACTCA    60GGCTGCT GCATTTGTTT CTTCATCATG GGTTTTCCAA    #CCCTTGTCAT   120ATGTTTC AGCTTGTGGA CACCTATGCT GCCTCCTATG    #GATTCTGTGA   180GAGCTCG TGGGGATCTC TTATGTGTAT GGCTTGCAAA    #GCTGGGCATT   240ATGATTG GATTCCAGCC TAACATCTTC TGGAAAGTCT    #GGGAACCCAT   300ATTTTAA CCTTTATCCT TTGCTTCAGC TTTTACCAGT    #TAATGCTCGC   360TACCGCT ATCCTAACTG GTCCATGGTG CTCGGATGGC    #CCCCTGGAAG   420TGGATCC CAATTATGTC TGTGATAAAA ATGCATCTGG    #CATTCTTAGC   480CTGAAGT TGGTGTGCTC GCCACAGCCG GACTGGGGCC    #CTTCCTTGGG   540GAGCGTT ACAAGAACAT GATCGACCCC TTGGGAACCT    #              589TGAAGG ATTTGGAACT GGGCACTCAG TGCTAGTCC    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 194 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #Phe Ile Met Gly Phe Proys Cys Ile Cys Phe    #                 15    #Gln Leu Val Asp Thr Tyrly Ile Tyr Met Phe    #             30    #Ile Phe Glu Leu Val Glyeu Val Ile Ile Ala    #         45    #Cys Glu Asp Ile Glu Metly Leu Gln Arg Phe    #     60    #Lys Val Cys Trp Ala Phero Asn Ile Phe Trp    # 80    #Cys Phe Ser Phe Tyr Glneu Thr Phe Ile Leu    #                 95    #Tyr Pro Asn Trp Ser Metyr Gly Ser Tyr Arg    #            110    #Val Ile Trp Ile Pro Ileet Leu Ala Cys Ser    #        125    #Gly Arg Phe Ile Glu Arget His Leu Ala Pro    #    140    #Trp Gly Pro Phe Leu Alaer Pro Gln Pro Asp    #160    #Ile Asp Pro Leu Gly Thrrg Tyr Lys Asn Met    #                175    #Asp Leu Glu Leu Gly Thrys Leu Pro Val Lys    #            190    -  Gln Cys    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2403 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    #GGAGGCAACG    60CTCCCAA GGAAATGAAT AAACCACCAA CCAACATCTT    #GGATCTTCCT   120GGGATAG CCCTCGAGCA CCTAGGACCA GCCCTGAGCA    #TTCCACCGGC   180CGGCCGC TGTCCAGCCG CCACGTGTGC CCAGGTCGGC    #TGGAGTAGGG   240AGTCTGC GGATGCGAGA GCCTGTGAGG CACAGCGGCC    #GGACTTAAGC   300GCAGCCC CCAGGCACAA GCGACCTCTG CGGCCCTCCG    #CAACGCTTTA   360CACAGGC CAATCCCCCT TCCGGGGCCG CTGGGGCTGG    #TGTGGCCAAG   420CAGCTCT GCGTGGCCCG GAGGAGGACG AGAACGTGAG    #CCAGAGCACA   480ACAACAA TACCCCACCC GTGGGCTGGG TGAATATGAG    #CACCACTACC   540CCGATGG AATCGCGTCG GTGCTCCCGG GCAGCGTGGC    #CAGCAAACTG   600AGCAAGG GGATGAGAAT AAGGCCAGAG GGAACTGGTC    #GAGGTTTCCC   660CCATGGT GGGGTACGCA GTGGGGCTGG GTAATGTTTG    #GATGCTGGCA   720AGAACGG GGGAGGTGCT TTCCTCATCC CTTACTTGAT    #CAGCCAGGGT   780CTATCTT CTTCCTAGAG GTGTCCCTGG GCCAGTTTGC    #GATGCTCATC   840GGAAGGC CATCCCAGCT CTGCAGGGCT GTGGCATTGC    #CTTCTACCTG   900TAGCCAT CTACTACAAC GTCATCATCT GCTACACGCT    #GAACACACCA   960TGTCTGT GCTGCCCTGG GGATCCTGCA ACAACCCGTG    #CCATCCCAAG  1020AAACCAA ACTTTTACTA GATTCCTGTG TTATCGGTGA    #CATGGTTAAC  1080ACTCTAC TTTCTGCATG ACTGCCTATC CGAACTTGAC    #CAAGTACTTT  1140CCAATAA GACATTTGTC AGCGGGAGTG AAGAGTACTT    #CTTGCCGTTC  1200CTGCAGG GATTGAATAT CCTGGTGAGA TCAGGTGGCC    #TAAGACATCA  1260CCTGGGT GATTGTATAT GCATCGCTGG CAAAAGGAAT    #CCTCCTCATT  1320ACTTCAC AGCCACCTTC CCTTATGTCG TCCTGGTCAT    #ACCTAAGTGG  1380TGCCTGG AGCTGGAGCC GGTATCTGGT ACTTCATCAC    #CTTCTCCCTG  1440ATGCCAC GGTGTGGAAG GATGCAGCCA CTCAGATTTT    #TAACAACTGC  1500GAGGGCT CATCACTCTT TCTTCTTACA ACAAATTCCA    #CGCTGGGTTT  1560TAATTGT AACCTGCACC AACAGTGCCA CTAGCATCTT    #TGAGAATGTG  1620TCATTGG CTTCATGGCC AACGAGCGCA AAGTCAACAT    #CAGGCTGCCT  1680CAGGCAT TGCATTTGTG GTTTACCCAG AAGCCTTAAC    #ACTTGACACC  1740GGGCCAT CATCTTTTTC CTGATGCTTC TCACGCTTGG    #CAAGTATCTG  1800TCGAGAC CATTGTGACC TCCATCTCGG ATGAGTTTCC    #TATGGGCTTC  1860CTGTGTT CACCCTGGGC TGCTGCATCT GCTTCTTCAT    #TGCTGCCTCC  1920AGGGTGG AATCTACATG TTTCAGCTTG TGGACACCTA    #GTACGGCTTG  1980TCATTGC CATATTTGAG CTTGTTGGCA TCTCCTATGT    #TTTCTGGAAG  2040AAGACAT CGAGATGATG ATTGGATTCC AGCCCAACAT    #CAGCTTCTAT  2100TTGTCAC ACCGACCATT TTAACGTTTA TCCTTTGCTT    #GGTGCTTGGA  2160TGACCTA TGGCTCCTAC CGCTACCCTA ACTGGTCCAT    #AAAAATGTAT  2220CCTGCTC CGTGATCTGG ATCCCGATTA TGTTCGTGAT    #GCCGGACTGG  2280GATTTAT TGAGAGGCTG AAGTTGGTAT GCTCGCCACA    #CCCCTTGGGA  2340CTCAGCA CCGCGGGGAA CGCTACAAGA ATATGATCGA    #CCAGTGCTAG  2400GACTCAA GCTGCCAGTG AAGGATTTGG AACTGGGCAC    #           2403    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 799 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #Lys Pro Pro Thr Asn Ilero Lys Glu Met Asn    #                 15    #Ser Pro Arg Ala Pro Argro Gly His Arg Asp    #             30    #Ala Pro Ala Ala Ala Valsp Leu Pro Ala Ala    #         45    #Thr Gly Ala Gln Thr Phero Arg Ser Ala Ser    #     60    #Gln Arg Pro Gly Val Glyrg Ala Cys Glu Ala    # 80    #Ala Thr Ser Ala Ala Leuer Pro Gln Ala Gln    #                 95    #Ala Asn Pro Pro Ser Glyly His Ser Ala Gln    #            110    #Lys Ile Pro Ala Leu Argsn Ala Leu His Cys    #        125    #Ala Lys Gly Thr Leu Glulu Asn Val Ser Val    #    140    #Asn Met Ser Gln Ser Thrro Val Gly Trp Val    #160    #Val Leu Pro Gly Ser Valsp Gly Ile Ala Ser    #                175    #Gly Asp Glu Asn Lys Alaro Glu Asp Glu Gln    #            190    #Ile Leu Ser Met Val Glyer Lys Leu Asp Phe    #        205    #Phe Pro Tyr Leu Ala Phely Asn Val Trp Arg    #    220    #Tyr Leu Met Met Leu Alala Phe Leu Ile Pro    #240    #Val Ser Leu Gly Gln Phele Phe Phe Leu Glu    #                255    #Ala Ile Pro Ala Leu Glnal Ser Val Trp Lys    #            270    #Val Leu Ile Ala Ile Tyret Leu Ile Ile Ser    #        285    #Tyr Leu Phe Ala Ser Pheys Tyr Thr Leu Phe    #    300    #Asn Pro Trp Asn Thr Prorp Gly Ser Cys Asn    #320    #Asp Ser Cys Val Ile Glyhr Lys Leu Leu Leu    #                335    #Thr Phe Cys Met Thr Alaln Ile Lys Asn Ser    #            350    #Ser Gln Ala Asn Lys Thret Val Asn Phe Thr    #        365    #Tyr Phe Val Leu Lys Ilelu Glu Tyr Phe Lys    #    380    #Arg Trp Pro Leu Pro Pheyr Pro Gly Glu Ile    #400    #Ala Ser Leu Ala Lys Glyrp Val Ile Val Tyr    #                415    #Thr Ala Thr Phe Pro Tyrys Val Val Tyr Phe    #            430    #Val Thr Leu Pro Gly Alaeu Leu Ile Arg Gly    #        445    #Lys Trp Glu Lys Leu Thryr Phe Ile Thr Pro    #    460    #Gln Ile Phe Phe Ser Leuys Asp Ala Ala Thr    #480    #Ser Ser Tyr Asn Lys Phely Leu Ile Thr Leu    #                495    #Val Thr Cys Thr Asn Serrg Asp Thr Leu Ile    #            510    #Phe Ser Val Ile Gly Phela Gly Phe Val Ile    #        525    #Asn Val Ala Asp Gln Glyys Val Asn Ile Glu    #    540    #Ala Leu Thr Arg Leu Proal Val Tyr Pro Glu    #560    #Leu Met Leu Leu Thr Leula Ile Ile Phe Phe    #                575    #Thr Ile Val Thr Ser Ilehe Ala Thr Ile Glu    #            590    #His Lys Pro Val Phe Thrys Tyr Leu Arg Thr    #        605    #Gly Phe Pro Met Ile Thrys Phe Phe Ile Met    #    620    #Asp Thr Tyr Ala Ala Seret Phe Gln Leu Val    #640    #Leu Val Gly Ile Ser Tyrle Ala Ile Phe Glu    #                655    #Ile Glu Met Met Ile Glyrg Phe Cys Glu Asp    #            670    #Trp Ala Phe Val Thr Prohe Trp Lys Val Cys    #        685    #Phe Tyr Gln Trp Glu Prole Leu Cys Phe Ser    #    700    #Trp Ser Met Val Leu Glyyr Arg Tyr Pro Asn    #720    #Ile Pro Ile Met Phe Valys Ser Val Ile Trp    #                735    #Ile Glu Arg Leu Lys Leula Pro Gly Arg Phe    #            750    #Phe Leu Ala Gln His Argro Asp Trp Gly Pro    #        765    #Leu Gly Thr Ser Ser Leusn Met Ile Asp Pro    #    780    #Leu Gly Thr Gln Cysro Val Lys Asp Leu Glu    #795    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    # 20               TTRTA    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    #                23AARCC NCC    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    #                23ACTTT NCC    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    #                24CACRA ANGC    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    #           30     TCCGC ATCAGACATG    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    # 20               CGCTG    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 33 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    #         33       CGTGA TCCTCCTCAT CCG    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    #             27   ATNGG RAANCCC    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    #          32      GAYYA RCAYTGNGTN CC    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    #           30     TTATC TGCTGCATGG    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 29 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    #            29    TTRTA NCKYTCNCC    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    #                23CCACA AGC    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    #               25 CACAC AAGCC    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 28 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    #             28   CATCC ACACTACT    __________________________________________________________________________

In summary, the sequences of the Sequences Listing are as follows:

    ______________________________________    SEQ ID  Type        Sequence   Corres Clone    ______________________________________    1       N.A.        nt 1-190   phG2-3-a    2       Protein     aa 1-63    3       N.A.        nt 1-190   phG2-3-b    4       Protein     aa 1-63    5       N.A..       nt 39-1254 phG2-1    6       Protein     aa 14-418    7       N.A.        nt 39-1635 phG2-2    8       Protein     aa 14-190    9       Protein     aa 192-545    10      N.A.        nt 1317-1847                                   phG2-4-a    11      Protein     aa 440-615    12      N.A.        nt 1317-1847                                   phG2-4-b    13      Protein     aa 440-615    14      N.A.        nt 1540-2379                                   phG2-7-a    15      Protein     aa 514-793    16      N.A.        nt 1540-2379                                   phG2-7-b    17      Protein     aa 514-793    18      N.A.        nt 1-2397    19      Protein     aa 1-797    20      N.A.        nt 1-2397  pHGT2    21      Protein     aa 1-797    22      N.A.        nt 1809-2397                                   phG2-8-a    23      Protein     aa 604-797    24      N.A.        nt 1809-2397                                   phG2-8-b    25      Protein     aa 604-797    ______________________________________

The nucleic acid sequences described herein, and consequently theprotein sequences derived therefrom, have been carefully sequenced.However, those of ordinary skill will recognize that nucleic acidsequencing technology can be susceptable to some error. Those ofordinary skill in the relevant arts are capable of validating orcorrecting these sequences based on the ample description herein ofmethods of isolating the nucleic acid sequences in question, and suchmodifications that are made readily available by the present disclosureare encompassed by the present invention. Furthermore, those sequencesreported herein are within the invention whether or not later clarifyingstudies identify sequencing errors.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred devices and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims that follow.

What is claimed:
 1. An isolated nucleic acid encoding a glycinetransporter comprising a sequence encoding the protein sequence of (i)SEQ ID 19 or (ii) a sequence corresponding to the protein sequence ofSEQ ID 19 except that it has one or more of the following amino acidsubstitutions (1) Ser¹⁰² to Gly, (2) Ser¹²⁴ to Phe, (3) Ile²⁷⁹ to Asn,(4) Arg³⁹³ to Gly, (5) Lys⁴⁵⁷ to Asn, (6) Asp⁴⁶³ to Asn, (7) Cys⁶¹⁰ toTyr, (8) Ile⁶¹¹ to Val, (9) Phe⁷³³ to Ser or (10) Ile⁷³⁵ to Val.
 2. Thenucleic acid of claim 1, comprising nucleotides 1-2391 of SEQ ID 18 orwith a sequence that varies from nucleotides 1-2391 of SEQ ID 18 byhaving one or more of the following nucleotide substitutions (a) T⁶ toC, (b) A³⁰⁴ to G, (c) C³⁷¹ to T, (d) C⁵⁷¹ to T, (e) T⁸³⁶ to A, (f) A¹¹¹⁶to G, (g) A¹¹⁷⁷ to G, (h) G¹³⁷¹ to C, (i) G¹³⁸⁷ to A, (j) G¹⁸²⁹ A, (k)A¹⁸³¹ to G, (l) G²¹⁰³ to A, (m) T²¹⁹⁸ to C, or (n) A²²⁰³ to G.
 3. Avector comprising the nucleic acid of claim 1 and an extrinsic promoterfunctionally associated therewith.
 4. A transformed cell that expressesa recombinant glycine transporter, comprising a vector that comprisesthe nucleic acid of claim
 1. 5. A method of producing a glycinetransporter comprising growing the cells of claim 4 to produce theglycine transporter.
 6. The method of claim 5 further comprising atleast one of (a) isolating membranes from said cells, which membranescomprise the glycine transporter or (b) extracting a protein fractionfrom the cells which fraction comprises the glycine transporter.